The PRESIDE (PhaRmacogEnomicS In DEpression) Trial: a double-blind randomised controlled trial of pharmacogenomic-informed prescribing of antidepressants on depression outcomes in patients with major depressive disorder in primary care.

BACKGROUND: The evidence for the clinical utility of pharmacogenomic (PGx) testing is growing, and guidelines exist for the use of PGx testing to inform prescribing of 13 antidepressants. Although previous randomised controlled trials of PGx testing for antidepressant prescribing have shown an association with remission of depression in clinical psychiatric settings, few trials have focused on the primary care setting, where most antidepressant prescribing occurs. METHODS: The PRESIDE Trial is a stratified double-blinded randomised controlled superiority trial that aims to evaluate the impact of a PGx-informed antidepressant prescribing report (compared with standard prescribing using the Australian Therapeutic Guidelines) on depressive symptoms after 12 weeks, when delivered in primary care. Six hundred seventy-two patients aged 18-65 years of general practitioners (GPs) in Victoria with moderate to severe depressive symptoms, measured using the Patient Health Questionnaire-9 (PHQ-9), will be randomly allocated 1:1 to each arm using a computer-generated sequence. Participants and GPs will be blinded to the study arm. The primary outcome is a difference between arms in the change of depressive symptoms, measured using the PHQ-9 after 12 weeks. Secondary outcomes include a difference between the arms in change in PHQ-9 score at 4, 8 and 26 weeks, proportion in remission at 12 weeks, a change in side effect profile of antidepressant medications, adherence to antidepressant medications, change in quality of life and cost-effectiveness of the intervention. DISCUSSION: This trial will provide evidence as to whether PGx-informed antidepressant prescribing is clinically efficacious and cost-effective. It will inform national and international policy and guidelines about the use of PGx to select antidepressants for people with moderate to severe depressive symptoms presenting in primary care. TRIAL REGISTRATION: Australian and New Zealand Clinical Trial Registry ACTRN12621000181808. Registered on 22 February 2021.

Neuroprotection of retinal ganglion cells by the sigma-1 receptor agonist pridopidine in models of experimental glaucoma.

Optic neuropathies such as glaucoma are characterized by retinal ganglion cell (RGC) degeneration and death. The sigma-1 receptor (S1R) is an attractive target for treating optic neuropathies as it is highly expressed in RGCs, and its absence causes retinal degeneration. Activation of the S1R exerts neuroprotective effects in models of retinal degeneration. Pridopidine is a highly selective and potent S1R agonist in clinical development. We show that pridopidine exerts neuroprotection of retinal ganglion cells in two different rat models of glaucoma. Pridopidine strongly binds melanin, which is highly expressed in the retina. This feature of pridopidine has implications to its ocular distribution, bioavailability, and effective dose. Mitochondria dysfunction is a key contributor to retinal ganglion cell degeneration. Pridopidine rescues mitochondrial function via activation of the S1R, providing support for the potential mechanism driving its neuroprotective effect in retinal ganglion cells.

The Sigma-1 Receptor Mediates Pridopidine Rescue of Mitochondrial Function in Huntington Disease Models.

Pridopidine is a selective Sigma-1 receptor (S1R) agonist in clinical development for Huntington disease (HD) and amyotrophic lateral sclerosis. S1R is a chaperone protein localized in mitochondria-associated endoplasmic reticulum (ER) membranes, a signaling platform that regulates Ca2+ signaling, reactive oxygen species (ROS) and mitochondrial fission. Here, we investigate the protective effects of pridopidine on various mitochondrial functions in human and mouse HD models. Pridopidine effects on mitochondrial dynamics were assessed in primary neurons from YAC128 HD mice expressing the mutant human HTT gene. We observe that pridopidine prevents the disruption of mitochondria-ER contact sites and improves the co-localization of inositol 1,4,5-trisphosphate receptor (IP3R) and its chaperone S1R with mitochondria in YAC128 neurons, leading to increased mitochondrial activity, elongation, and motility. Increased mitochondrial respiration is also observed in YAC128 neurons and in pridopidine-treated HD human neural stem cells (hNSCs). ROS levels were assessed after oxidative insult or S1R knockdown in pridopidine-treated YAC128 neurons, HD hNSCs, and human HD lymphoblasts. All HD models show increased ROS levels and deficient antioxidant response, which are efficiently rescued with pridopidine. Importantly, pridopidine treatment before H2O2-induced mitochondrial dysfunction and S1R presence are required for HD cytoprotection. YAC128 mice treated at early/pre-symptomatic age with pridopidine show significant improvement in motor coordination, indicating a delay in symptom onset. Additionally, in vivo pridopidine treatment reduces mitochondrial ROS levels by normalizing mitochondrial complex activity. In conclusion, S1R-mediated enhancement of mitochondrial function contributes to the neuroprotective effects of pridopidine, providing insight into its mechanism of action and therapeutic potential.

PTPα is required for laminin-2-induced Fyn-Akt signaling to drive oligodendrocyte differentiation.

Extrinsic signals that regulate oligodendrocyte maturation and subsequent myelination are essential for central nervous system development and regeneration. Deficiency in the extracellular factor laminin-2 (Lm2, comprising the α2ß1γ1 chains), as occurs in congenital muscular dystrophy, can lead to impaired oligodendroglial development and aberrant myelination, but many aspects of Lm2-regulated oligodendroglial signaling and differentiation remain undefined. We show that receptor-like protein tyrosine phosphatase α (PTPα, also known as PTPRA) is essential for myelin basic protein expression and cell spreading during Lm2-induced oligodendrocyte differentiation. PTPα complexes with the Lm2 receptors α6ß1 integrin and dystroglycan to transduce Fyn activation upon Lm2 engagement. In this way, PTPα mediates a subset of Lm2-induced signals required for differentiation, includeing mTOR-dependent Akt activation but not Erk1/2 activation. We identify N-myc downstream regulated gene-1 (NDRG1) as a PTPα-regulated molecule during oligodendrocyte differentiation, and distinguish Lm2 receptor-specific modes of Fyn-Akt-dependent and -independent NDRG1 phosphorylation. Altogether, this reveals an Lm2-regulated PTPα-Fyn-Akt signaling axis that is critical for key aspects of the gene expression and morphological changes that mark oligodendrocyte maturation.

Glial and Neuronal Protein Tyrosine Phosphatase Alpha (PTPα) Regulate Oligodendrocyte Differentiation and Myelination.

CNS myelination defects occur in mice deficient in receptor-like protein tyrosine phosphatase alpha (PTPα). Here, we investigated the role of PTPα in oligodendrocyte differentiation and myelination using cells and tissues from wild-type (WT) and PTPα knockout (KO) mice. PTPα promoted the timely differentiation of neural stem cell-derived oligodendrocyte progenitor cells (OPCs). Compared to WT OPCs, KO OPC cultures had more NG2+ progenitors, fewer myelin basic protein (MBP)+ oligodendrocytes, and reduced morphological complexity. In longer co-cultures with WT neurons, more KO than WT OPCs remained NG2+ and while equivalent MBP+ populations of WT and KO cells formed, the reduced area occupied by the MBP+ KO cells suggested that their morphological maturation was impeded. These defects were associated with reduced myelin formation in KO OPC/WT neuron co-cultures. Myelin formation was also impaired when WT OPCs were co-cultured with KO neurons, revealing a novel role for neuronal PTPα in myelination. Canonical Wnt/ß-catenin signaling is an important regulator of OPC differentiation and myelination. Wnt signaling activity was not dysregulated in OPCs lacking PTPα, but suppression of Wnt signaling by the small molecule XAV939 remediated defects in KO oligodendrocyte differentiation and enhanced myelin formation by KO oligodendrocytes. However, the myelin segments that formed were significantly shorter than those produced by WT oligodendrocytes, raising the possibility of a role for glial PTPα in myelin extension distinct from its pro-differentiating actions. Altogether, this study reveals PTPα as a molecular coordinator of oligodendroglial and neuronal signals that controls multiple aspects of oligodendrocyte development and myelination.

Down-regulation of MIF by NFκB under hypoxia accelerated neuronal loss during stroke.

Neuronal apoptosis is one of the major causes of poststroke neurological deficits. Inflammation during the acute phase of stroke results in nuclear translocation of NFκB in affected cells in the infarct area. Macrophage migration inhibitory factor (MIF) promotes cardiomyocyte survival in mice following heart ischemia. However, the role of MIF during stroke remains limited. In this study, we showed that MIF expression is down-regulated by 0.75 ± 0.10-fold of the control in the infarct area in the mouse brains. Two functional cis-acing NFκB response elements were identified in the human MIF promoter. Dual activation of hypoxia and NFκB signaling resulted in significant reduction of MIF promoter activity to 0.86 ± 0.01-fold of the control. Furthermore, MIF reduced caspase-3 activation and protected neurons from oxidative stress- and in vitro ischemia/reperfusion-induced apoptosis. H2O2 significantly induced cell death with 12.81 ± 0.58-fold increase of TUNEL-positive cells, and overexpression of MIF blocked the H2O2-induced cell death. Disruption of the MIF gene in MIF-knockout mice resulted in caspase-3 activation, neuronal loss, and increased infarct development during stroke in vivo. The infarct volume was increased from 6.51 ± 0.74% in the wild-type mice to 9.07 ± 0.66% in the MIF-knockout mice. Our study demonstrates that MIF exerts a neuronal protective effect and that down-regulation of MIF by NFκB-mediated signaling under hypoxia accelerates neuronal loss during stroke. Our results suggest that MIF is an important molecule for preserving a longer time window for stroke treatment, and strategies to maintain MIF expression at physiological level could have beneficial effects for stroke patients.

Aberrant expression of RCAN1 in Alzheimer's pathogenesis: a new molecular mechanism and a novel drug target.

AD, a devastating neurodegenerative disorder, is the most common cause of dementia in the elderly. Patients with AD are characterized by three hallmarks of neuropathology including neuritic plaque deposition, neurofibrillary tangle formation, and neuronal loss. Growing evidences indicate that dysregulation of regulator of calcineurin 1 (RCAN1) plays an important role in the pathogenesis of AD. Aberrant RCAN1 expression facilitates neuronal apoptosis and Tau hyperphosphorylation, leading to neuronal loss and neurofibrillary tangle formation. This review aims to describe the recent advances of the regulation of RCAN1 expression and its physiological functions. Moreover, the AD risk factors-induced RCAN1 dysregulation and its role in promoting neuronal loss, synaptic impairments and neurofibrillary tangle formation are summarized. Furthermore, we provide an outlook into the effects of RCAN1 dysregulation on APP processing, Aß generation and neuritic plaque formation, and the possible underlying mechanisms, as well as the potential of targeting RCAN1 as a new therapeutic approach.

Inhibition of GSK3ß-mediated BACE1 expression reduces Alzheimer-associated phenotypes.

Deposition of amyloid ß protein (Aß) to form neuritic plaques in the brain is the pathological hallmark of Alzheimer's disease (AD). Aß is generated from sequential cleavages of the ß-amyloid precursor protein (APP) by the ß- and γ-secretases, and ß-site APP-cleaving enzyme 1 (BACE1) is the ß-secretase essential for Aß generation. Previous studies have indicated that glycogen synthase kinase 3 (GSK3) may play a role in APP processing by modulating γ-secretase activity, thereby facilitating Aß production. There are two highly conserved isoforms of GSK3: GSK3α and GSK3ß. We now report that specific inhibition of GSK3ß, but not GSK3α, reduced BACE1-mediated cleavage of APP and Aß production by decreasing BACE1 gene transcription and expression. The regulation of BACE1 gene expression by GSK3ß was dependent on NF-κB signaling. Inhibition of GSK3 signaling markedly reduced Aß deposition and neuritic plaque formation, and rescued memory deficits in the double transgenic AD model mice. These data provide evidence for regulation of BACE1 expression and AD pathogenesis by GSK3ß and that inhibition of GSK3 signaling can reduce Aß neuropathology and alleviate memory deficits in AD model mice. Our study suggests that interventions that specifically target the ß-isoform of GSK3 may be a safe and effective approach for treating AD.

Sp1 regulates human huntingtin gene expression.

Huntington's disease (HD) is a hereditary neurodegenerative disorder resulting from the expansion of a polyglutamine tract in the huntingtin protein. The expansion of cytosine-adenine-guanine repeats results in neuronal loss in the striatum and cortex. Mutant huntingtin (HTT) may cause toxicity via a range of different mechanisms. Recent studies indicate that impairment of wild-type HTT function may also contribute to HD pathogenesis. However, the mechanisms regulating HTT expression have not been well defined. In this study, we cloned 1,795 bp of the 5' flanking region of the human huntingtin gene (htt) and identified a 106-bp fragment containing the transcription start site as the minimal region necessary for promoter activity. Sequence analysis reveals several putative regulatory elements including Sp1, NF-κB, HIF, CREB, NRSF, P53, YY1, AP1, and STAT in the huntingtin promoter. We found functional Sp1 response elements in the huntingtin promoter region. The expression of Sp1 enhanced huntingtin gene transcription and the inhibition of Sp1-mediated transcriptional activation reduced huntingtin gene expression. These results suggest that Sp1 plays an important role in the regulation of the human huntingtin gene expression at the mRNA and protein levels. Our study suggests that the dysregulation of Sp1-mediated huntingtin transcription, combining with mutant huntingtin's detrimental effect on other Sp1-mediated downstream gene function, may contribute to the pathogenesis of HD.

Detection of neuritic plaques in Alzheimer's disease mouse model.

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer's disease. The central component of neuritic plaques is a small filamentous protein called amyloid ß protein (Aß)(1), which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by ß-secretase and γ-secretase. The amyloid hypothesis entails that Aγ-containing plaques as the underlying toxic mechanism in AD pathology(2). The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of Aγ neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.

NF-κB signaling inhibits ubiquitin carboxyl-terminal hydrolase L1 gene expression.

Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that plays a regulatory role in targeting proteins for proteasomal degradation. UCH-L1 is highly expressed in neurons and has been demonstrated to promote cell viability and maintain neuronal integrity. Reduced UCH-L1 levels have been observed in various neurodegenerative diseases, and expression of UCH-L1 can rescue synaptic dysfunction and memory deficits in Alzheimer's Disease model mice. However, the mechanisms regulating UCH-L1 expression have not been determined. In this study, we cloned a 1782 bp of the 5' flanking region of the human UCH-L1 gene and identified a 43 bp fragment containing the transcription start site as the minimal region necessary for promoter activity. Sequence analysis revealed several putative regulatory elements including NF-κB, NFAT, CREB, NRSF, YY1, AP1, and STAT in the UCH-L1 promoter. A functional NF-κB response element was identified in the UCH-L1 promoter region. Expression of NF-κB suppressed UCH-L1 gene transcription. In the RelA knockout system where NF-κB activity is ablated, UCH-L1 expression was significantly increased. Furthermore, activation of NF-κB signaling by the inflammatory stimulator lipopolysaccharide and TNFα resulted in a decrease of UCH-L1 gene expression by inhibiting its transcription. As NF-κB is an important signaling module in inflammatory response, our study suggests a possibility that inflammation might compromise neuronal functions via the interaction of NF-κB and UCH-L1. A better understanding of the NF-κB-regulated UCH-L1 transcription will provide insights to the role of inflammatory responses in Alzheimer's disease and Parkinson's disease.

PARADOXICAL RESPONSES TO NEUROTOXIC STERYL GLYCOSIDES: INSIGHTS FROM A CELLULAR MODEL OF ALSPDC.

The causes of many sporadic neurodegenerative diseases remain unknown making prevention difficult, if not impossible. One clue comes from the study of amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS-PDC) of Guam which shares many similarities with amyotrophic lateral sclerosis, Parkinson's disease, and Alzheimer's disease seen in other parts of the world. This disorder may provide a unique opportunity to study the cause and progression of neurodegenerative diseases. Epidemiological and experimental findings indicate that dietary consumption of cycad seeds is an underlying cause of ALS-PDC. Our laboratory provided evidence that a family of compounds called steryl glycosides are the active ingredients that may be responsible for producing the neurodegenerative outcome in ALS-PDC. Here, we review some of our work on the chronic toxicity of steryl glycosides in neuronal cells maintained in cell culture and in an in vivo mouse model. The current studies indicate some mechanisms about how neuronal cells respond to this class of toxins.

Valproic acid inhibits Abeta production, neuritic plaque formation, and behavioral deficits in Alzheimer's disease mouse models.

Neuritic plaques in the brains are one of the pathological hallmarks of Alzheimer's disease (AD). Amyloid beta-protein (Abeta), the central component of neuritic plaques, is derived from beta-amyloid precursor protein (APP) after beta- and gamma-secretase cleavage. The molecular mechanism underlying the pathogenesis of AD is not yet well defined, and there has been no effective treatment for AD. Valproic acid (VPA) is one of the most widely used anticonvulsant and mood-stabilizing agents for treating epilepsy and bipolar disorder. We found that VPA decreased Abeta production by inhibiting GSK-3beta-mediated gamma-secretase cleavage of APP both in vitro and in vivo. VPA treatment significantly reduced neuritic plaque formation and improved memory deficits in transgenic AD model mice. We also found that early application of VPA was important for alleviating memory deficits of AD model mice. Our study suggests that VPA may be beneficial in the prevention and treatment of AD.

Cholesteryl Glucoside Stimulates Activation of Protein Kinase B/Akt in the Motor Neuron-Derived NSC34 Cell Line.

Steryl glycosides and related compounds are commonly found in the environment and have been associated with neurodegenerative changes in vulnerable individuals. However, their mechanisms of action in mammalian cells have not been well investigated. In the present study the effects of cholesterol glucoside (CG), a variant form of steryl glycoside, was investigated in the motor neuron-derived NSC34 cell line. Prolonged treatment with CG was found to induce cell death in a dose- and time-dependent manner. However, transient exposure of CG preconditioned NSC34 cells for stress from serum deprivation. To study the signaling pathways activated by CG, we employed the Kinetworks™ KPSS 1.3 Phospho-site Screen to track the phosphorylation level of at least 35 diverse signaling proteins. The survival protein kinase B (PKB/Akt) displayed a 2-fold increase in phosphorylation at its Ser-473 activation site following CG stimulation. Akt signaling was important for conferring cytoprotection against serum deprivation-induced stress. Inhibition of phosphatidylinositol 3-kinase (PI3K), which indirectly triggers Akt stimulation, completely abolished CG preconditioning against serum deprivation. Our findings revealed that there may be a PI3K-independent pathway which also mediated Akt Ser-473 phosphorylation. Improved understanding of the mechanisms of action of CG should provide insights to the how other members of the steryl glycoside family induce toxicity in the mouse model of ALS-PDC, and how cells respond to these toxins.