Intranasal Administration of Forskolin and Noopept Reverses Parkinsonian Pathology in PINK1 Knockout Rats.

Parkinson's Disease (PD) is a brain-degenerative disorder characterized by a progressive loss of midbrain dopamine neurons. Current standard-of-care includes oral administration of Levodopa to address motor symptoms, but this treatment is not disease-modifying. A reduction in Protein Kinase A (PKA) signaling and neurotrophic support contributes to PD pathology. We previously showed that enhancing PKA activity in the brain via intraperitoneal administration of Forskolin in Parkinsonian rats (PINK1 knockout) abrogate motor symptoms and loss of midbrain dopamine neurons. Given that intraperitoneal administration is invasive, we hypothesized that intranasal administration of Forskolin and a second nootropic agent (Noopept) could reverse PD pathology efficiently. Results show that intranasal administration of a formulation (CNS/CT-001) containing Forskolin (10 µM) and Noopept (20 nM) significantly reversed motor symptoms, loss of hind limb strength, and neurodegeneration of midbrain dopamine neurons in PINK1-KO rats and is indistinguishable from wild-type (WT) rats; therapeutic effects associated with increased PKA activity and levels of BDNF and NGF in the brain. Intranasal administration of CNS/CT-001, but not Forskolin, significantly decreased the number of α-synuclein aggregates in the cortex of PINK1-KO rats, and is indistinguishable from WT rats. Overall, we show proof of concept that intranasal administration of CNS/CT-001 is a non-invasive, disease-modifying formulation for PD.

PINK1 regulates mitochondrial trafficking in dendrites of cortical neurons through mitochondrial PKA.

Mitochondrial Protein Kinase A (PKA) and PTEN-induced kinase 1 (PINK1), which is linked to Parkinson's disease, are two neuroprotective serine/threonine kinases that regulate dendrite remodeling and mitochondrial function. We have previously shown that PINK1 regulates dendrite morphology by enhancing PKA activity. Here, we show the molecular mechanisms by which PINK1 and PKA in the mitochondrion interact to regulate dendrite remodeling, mitochondrial morphology, content, and trafficking in dendrites. PINK1-deficient cortical neurons exhibit impaired mitochondrial trafficking, reduced mitochondrial content, fragmented mitochondria, and a reduction in dendrite outgrowth compared to wild-type neurons. Transient expression of wild-type, but not a PKA-binding-deficient mutant of the PKA-mitochondrial scaffold dual-specificity A Kinase Anchoring Protein 1 (D-AKAP1), restores mitochondrial trafficking, morphology, and content in dendrites of PINK1-deficient cortical neurons suggesting that recruiting PKA to the mitochondrion reverses mitochondrial pathology in dendrites induced by loss of PINK1. Mechanistically, full-length and cleaved forms of PINK1 increase the binding of the regulatory subunit ß of PKA (PKA/RIIß) to D-AKAP1 to enhance the autocatalytic-mediated phosphorylation of PKA/RIIß and PKA activity. D-AKAP1/PKA governs mitochondrial trafficking in dendrites via the Miro-2/TRAK2 complex and by increasing the phosphorylation of Miro-2. Our study identifies a new role of D-AKAP1 in regulating mitochondrial trafficking through Miro-2, and supports a model in which PINK1 and mitochondrial PKA participate in a similar neuroprotective signaling pathway to maintain dendrite connectivity.

Novel Redox-Dependent Esterase Activity (EC 3.1.1.2) for DJ-1: Implications for Parkinson's Disease.

Mutations the in human DJ-1 (hDJ-1) gene are associated with early-onset autosomal recessive forms of Parkinson's disease (PD). hDJ-1/parkinsonism associated deglycase (PARK7) is a cytoprotective multi-functional protein that contains a conserved cysteine-protease domain. Given that cysteine-proteases can act on both amide and ester substrates, we surmised that hDJ-1 possessed cysteine-mediated esterase activity. To test this hypothesis, hDJ-1 was overexpressed, purified and tested for activity towards 4-nitrophenyl acetate (pNPA) as µmol of pNPA hydrolyzed/min/mg·protein (U/mg protein). hDJ-1 showed maximum reaction velocity esterase activity (Vmax = 235.10 ± 12.00 U/mg protein), with a sigmoidal fit (S0.5 = 0.55 ± 0.040 mM) and apparent positive cooperativity (Hill coefficient of 2.05 ± 0.28). A PD-associated mutant of DJ-1 (M26I) lacked activity. Unlike its protease activity which is inactivated by reactive oxygen species (ROS), esterase activity of hDJ-1 is enhanced upon exposure to low concentrations of hydrogen peroxide (<10 µM) and plateaus at elevated concentrations (>100 µM) suggesting that its activity is resistant to oxidative stress. Esterase activity of DJ-1 requires oxidation of catalytic cysteines, as chemically protecting cysteines blocked its activity whereas an oxido-mimetic mutant of DJ-1 (C106D) exhibited robust esterase activity. Molecular docking studies suggest that C106 and L126 within its catalytic site interact with esterase substrates. Overall, our data show that hDJ-1 contains intrinsic redox-sensitive esterase activity that is abolished in a PD-associated mutant form of the hDJ-1 protein.

Intraperitoneal Administration of Forskolin Reverses Motor Symptoms and Loss of Midbrain Dopamine Neurons in PINK1 Knockout Rats.

BACKGROUND: Parkinson's disease (PD) is a relentless, chronic neurodegenerative disease characterized by the progressive loss of substantia nigra (SN) neurons that leads to the onset of motor and non-motor symptoms. Standard of care for PD consists of replenishing the loss of dopamine through oral administration of Levodopa; however, this treatment is not disease-modifying and often induces intolerable side effects. While the etiology that contributes to PD is largely unknown, emerging evidence in animal models suggests that a significant reduction in neuroprotective Protein Kinase A (PKA) signaling in the SN contributes to PD pathogenesis, suggesting that restoring PKA signaling in the midbrain may be a new anti-PD therapeutic alternative. OBJECTIVE: We surmised that pharmacological activation of PKA via intraperitoneal administration of Forskolin exerts anti-PD effects in symptomatic PTEN-induced kinase 1 knockout (PINK1-KO), a bona fide in vivo model of PD. METHODS: By using a beam balance and a grip strength analyzer, we show that Forskolin reverses motor symptoms and loss of hindlimb strength with long-lasting therapeutic effects (> 5 weeks) following the last dose. RESULTS: In comparison, intraperitoneal treatment with Levodopa temporarily (24 h) reduces motor symptoms but unable to restore hindlimb strength in PINK1-KO rats. By using immunohistochemistry and an XF24e BioAnalyzer, Forskolin treatment reverses SN neurons loss, elevates brain energy production and restores PKA activity in SN in symptomatic PINK1-KO rats. CONCLUSION: Overall, our collective in vivo data suggest that Forskolin is a promising disease-modifying therapeutic alternative for PD and is superior to Levodopa because it confers long-lasting therapeutic effects.

Blood mononuclear cells as speculum of emotional stress analyzed by synchrotron infrared spectroscopy and a nootropic drug.

Chronic psychological stress is an important public health issue which generates behavioral changes, anxiety, immunosuppression and oxidative damage. Piracetam is a cognitive enhancer, at cellular level it protects from oxidative stress. The aim of this study was to evaluate the effect of psychological stress and of piracetam on circulating mononuclear cells by analyzing the biochemical spectrome using Synchrotron Radiation Fourier Transform Infrared Microspectroscopy (SR-µFTIR). Rats were exposed for five days to a stressor (cat odor) under oral administration of piracetam (600 mg/kg). SR-µFTIR analysis showed a decrease in bands associated to the lipids region (2852 cm-1, 2923 cm-1 and 2962 cm-1) and an increase absorption of the amide I band (1654 cm-1) under stress conditions. The principal component analysis showed increase oxidation of lipids (decrease of 3010 cm-1, 2923 cm-1 and 2852 cm-1 bands) as well as proteins denaturation (increase of 1610 cm-1 and 1690 cm-1 bands) under stress. Piracetam provided protection to polyunsaturated lipids (p ≤ 0.001) and lipids/proteins ratio (p ≤ 0.001). Behaviorally, this drug diminished fear and anxiety in stressed animals by the plus maze test (p ≤ 0.002). However, this drug induced oxidative stress in mononuclear cells from unstressed animals and altered their behavior.