Glypican-4 serum levels are associated with cognitive dysfunction and vascular risk factors in Parkinson's disease

Glypicans are biomarkers for various pathologies, including cardiovascular disease, cancer and diabetes. Increasing evidence suggests that glypicans also play a role in the context of neurodegenerative disorders. Initially described as supporting functionality of synapses via glutamate receptors during CNS development, Glypican 4 (GPC-4) also plays a role in the context of dementia via tau hyperphosphorylation in Alzheimer's disease, which is also a co-pathology in Parkinson's disease dementia. However, clinical evidence of circulating GPC-4 in Parkinson's disease (PD) is missing so far. We therefore investigated GPC-4 in biofluids of PD patients. We analyzed GPC-4 levels in cerebrospinal fluid (CSF, n = 140), serum (n = 80), and tear fluid samples (n = 70) of PD patients and control subjects in a similar age range by ELISA (serum, CSF) and western blot (tear fluid). Expression of circulating GPC-4 was confirmed in all three biofluids, with highest levels in serum. Interestingly, GPC-4 levels were age-dependent, and multiple regression analysis revealed a significant association between GPC-4 serum levels and MoCA score, suggesting an involvement of GPC-4 in PD-associated cognitive decline. Furthermore, stratification of PD patients for vascular risk factors revealed a significant increase of GPC-4 serum levels in PD patients with vascular risk factors. Our results suggest GPC-4 as a clinical biomarker for vascular risk stratification in order to identify PD patients with increased risk of developing dementia

AAV-mediated inhibition of ULK1 promotes axonal regeneration in the central nervous system in vitro and in vivo

Axonal damage is an early step in traumatic and neurodegenerative disorders of the central nervous system (CNS). Damaged axons are not able to regenerate sufficiently in the adult mammalian CNS, leading to permanent neurological deficits. Recently, we showed that inhibition of the autophagic protein ULK1 promotes neuroprotection in different models of neurodegeneration. Moreover, we demonstrated previously that axonal protection improves regeneration of lesioned axons. However, whether axonal protection mediated by ULK1 inhibition could also improve axonal regeneration is unknown. Here, we used an adeno-associated viral (AAV) vector to express a dominant-negative form of ULK1 (AAV.ULK1.DN) and investigated its effects on axonal regeneration in the CNS. We show that AAV.ULK1.DN fosters axonal regeneration and enhances neurite outgrowth in vitro. In addition, AAV.ULK1.DN increases neuronal survival and enhances axonal regeneration after optic nerve lesion, and promotes long-term axonal protection after spinal cord injury (SCI) in vivo. Interestingly, AAV.ULK1.DN also increases serotonergic and dopaminergic axon sprouting after SCI. Mechanistically, AAV.ULK1.DN leads to increased ERK1 activation and reduced expression of RhoA and ROCK2. Our findings outline ULK1 as a key regulator of axonal degeneration and regeneration, and define ULK1 as a promising target to promote neuroprotection and regeneration in the CNS

Fasudil attenuates aggregation of α-synuclein in models of Parkinson’s disease

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, yet disease-modifying treatments do not currently exist. Rho-associated protein kinase (ROCK) was recently described as a novel neuroprotective target in PD. Since alpha-synuclein (alpha-Syn) aggregation is a major hallmark in the pathogenesis of PD, we aimed to evaluate the anti-aggregative potential of pharmacological ROCK inhibition using the isoquinoline derivative Fasudil, a small molecule inhibitor already approved for clinical use in humans. Fasudil treatment significantly reduced alpha-Syn aggregation in vitro in a H4 cell culture model as well as in a cell-free assay. Nuclear magnetic resonance spectroscopy analysis revealed a direct binding of Fasudil to tyrosine residues Y133 and Y136 in the C-terminal region of alpha-Syn. Importantly, this binding was shown to be biologically relevant using site-directed mutagenesis of these residues in the cell culture model. Furthermore, we evaluated the impact of long-term Fasudil treatment on alpha-Syn pathology in vivo in a transgenic mouse model overexpressing human alpha-Syn bearing the A53T mutation (alpha-Syn(A53T) mice). Fasudil treatment improved motor and cognitive functions in alpha-Syn(A53T) mice as determined by Catwalk (TM) gait analysis and novel object recognition (NOR), without apparent side effects. Finally, immunohistochemical analysis revealed a significant reduction of alpha-Syn pathology in the midbrain of alpha-Syn(A53T) mice after Fasudil treatment. Our results demonstrate that Fasudil, next to its effects mediated by ROCK-inhibition, directly interacts with alpha-Syn and attenuates alpha-Syn pathology. This underscores the translational potential of Fasudil as a disease-modifying drug for the treatment of PD and other synucleinopathies

Proteomic analysis of the human hippocampus identifies neuronal pentraxin 1 (NPTX1) as synapto-axonal target in late-stage Parkinson's disease

Parkinson's disease (PD) affects a significant proportion of the population over the age of 60 years, and its prevalence is increasing. While symptomatic treatment is available for motor symptoms of PD, non-motor complications such as dementia result in diminished life quality for patients and are far more difficult to treat. In this study, we analyzed PD-associated alterations in the hippocampus of PD patients, since this brain region is strongly affected by PD dementia. We focused on synapses, analyzing the proteome of post-mortal hippocampal tissue from 16 PD cases and 14 control subjects by mass spectrometry. Whole tissue lysates and synaptosomal fractions were analyzed in parallel. Differential analysis combined with bioinformatic network analyses identified neuronal pentraxin 1 (NPTX1) to be significantly dysregulated in PD and interacting with proteins of the synaptic compartment. Modulation of NPTX1 protein levels in primary hippocampal neuron cultures validated its role in synapse morphology. Our analysis suggests that NPTX1 contributes to synaptic pathology in late-stage PD and represents a putative target for novel therapeutic strategies

Glypican-4 serum levels are associated with cognitive dysfunction and vascular risk factors in Parkinson’s disease

Abstract Glypicans are biomarkers for various pathologies, including cardiovascular disease, cancer and diabetes. Increasing evidence suggests that glypicans also play a role in the context of neurodegenerative disorders. Initially described as supporting functionality of synapses via glutamate receptors during CNS development, Glypican 4 (GPC-4) also plays a role in the context of dementia via tau hyperphosphorylation in Alzheimer’s disease, which is also a co-pathology in Parkinson’s disease dementia. However, clinical evidence of circulating GPC-4 in Parkinson’s disease (PD) is missing so far. We therefore investigated GPC-4 in biofluids of PD patients. We analyzed GPC-4 levels in cerebrospinal fluid (CSF, n = 140), serum (n = 80), and tear fluid samples (n = 70) of PD patients and control subjects in a similar age range by ELISA (serum, CSF) and western blot (tear fluid). Expression of circulating GPC-4 was confirmed in all three biofluids, with highest levels in serum. Interestingly, GPC-4 levels were age-dependent, and multiple regression analysis revealed a significant association between GPC-4 serum levels and MoCA score, suggesting an involvement of GPC-4 in PD-associated cognitive decline. Furthermore, stratification of PD patients for vascular risk factors revealed a significant increase of GPC-4 serum levels in PD patients with vascular risk factors. Our results suggest GPC-4 as a clinical biomarker for vascular risk stratification in order to identify PD patients with increased risk of developing dementia

Sensory Axon growth requires spatiotemporal integration of CaSR and TrkB Signaling

Neural circuit development involves the coordinated growth and guidance of axons. During this process, axons encounter many different cues, but how these cues are integrated and translated into growth is poorly understood. In this study, we report that receptor signaling does not follow a linear path but changes dependent on developmental stage and coreceptors involved. Using developing chicken embryos of both sexes, our data show that calcium-sensing receptor (CaSR), a G-protein-coupled receptor important for regulating calcium homeostasis, regulates neurite growth in two distinct ways. First, when signaling in isolation, CaSR promotes growth through the PI3-kinase-Akt pathway. At later developmental stages, CaSR enhances tropomyosin receptor kinase B (TrkB)/BDNF-mediated neurite growth. This enhancement is facilitated through a switch in the signaling cascade downstream of CaSR (i.e., from the PI3-kinase-Akt pathway to activation of GSK3α Tyr279). TrkB and CaSR colocalize within late endosomes, cotraffic and coactivate GSK3, which serves as a shared signaling node for both receptors. Our study provides evidence that two unrelated receptors can integrate their individual signaling cascades toward a nonadditive effect and thus control neurite growth during development. SIGNIFICANCE STATEMENT This work highlights the effect of receptor coactivation and signal integration in a developmental setting. During embryonic development, neurites grow toward their targets guided by cues in the extracellular environment. These cues are sensed by receptors at the surface that trigger intracellular signaling events modulating the cytoskeleton. Emerging evidence suggests that the effects of guidance cues are diversified, therefore expanding the number of responses. Here, we show that two unrelated receptors can change the downstream signaling cascade and regulate neuronal growth through a shared signaling node. In addition to unraveling a novel signaling pathway in neurite growth, this research stresses the importance of receptor coactivation and signal integration during development of the nervous system

Sensory Axon growth requires spatiotemporal integration of CaSR and TrkB Signaling

Neural circuit development involves the coordinated growth and guidance of axons. During this process, axons encounter many different cues, but how these cues are integrated and translated into growth is poorly understood. In this study, we report that receptor signaling does not follow a linear path but changes dependent on developmental stage and coreceptors involved. Using developing chicken embryos of both sexes, our data show that calcium-sensing receptor (CaSR), a G-protein-coupled receptor important for regulating calcium homeostasis, regulates neurite growth in two distinct ways. First, when signaling in isolation, CaSR promotes growth through the PI3-kinase-Akt pathway. At later developmental stages, CaSR enhances tropomyosin receptor kinase B (TrkB)/BDNF-mediated neurite growth. This enhancement is facilitated through a switch in the signaling cascade downstream of CaSR (i.e., from the PI3-kinase-Akt pathway to activation of GSK3α Tyr279). TrkB and CaSR colocalize within late endosomes, cotraffic and coactivate GSK3, which serves as a shared signaling node for both receptors. Our study provides evidence that two unrelated receptors can integrate their individual signaling cascades toward a nonadditive effect and thus control neurite growth during development. SIGNIFICANCE STATEMENT This work highlights the effect of receptor coactivation and signal integration in a developmental setting. During embryonic development, neurites grow toward their targets guided by cues in the extracellular environment. These cues are sensed by receptors at the surface that trigger intracellular signaling events modulating the cytoskeleton. Emerging evidence suggests that the effects of guidance cues are diversified, therefore expanding the number of responses. Here, we show that two unrelated receptors can change the downstream signaling cascade and regulate neuronal growth through a shared signaling node. In addition to unraveling a novel signaling pathway in neurite growth, this research stresses the importance of receptor coactivation and signal integration during development of the nervous system

Additional file 2: Figure S2. of Fasudil attenuates aggregation of α-synuclein in models of Parkinson’s disease

Blood cell counts and biochemical analysis of renal and hepatic function parameters in α-SynA53T mice. a Leucocytes (LEU). b Erythrocytes (ERY). c Thrombocytes (THR). d Hemoglobin (HGB). e Hematocrit (HCT). f Creatinine (CREA). g Alanine aminotransferase (ALT). h Aspartate aminotransferase (AST). i gamma-glutamyl transferase (GGT, # = all values below detection level). All measured parameters were in the normal range for laboratory mice. a-e: n(wt ctrl) = 5, n(A53T ctrl) = 2, n(A53T Fas10) = 4, n(A53T Fas30) = 5; f-i: n(wt ctrl) = 5, n(A53T ctrl) = 1, n(A53T Fas10) = 8, n(A53T Fas30) = 7. (TIF 125 kb