Substantia Nigra Pars Reticulata Projections to the Pedunculopontine Nucleus Modulate Dyskinesia.

BACKGROUND: Long-term use of levodopa for Parkinson's disease (PD) treatment is often hindered by development of motor complications, including levodopa-induced dyskinesia (LID). The substantia nigra pars reticulata (SNr) and globus pallidus internal segment (GPi) are the output nuclei of the basal ganglia. Dysregulation of SNr and GPi activity contributes to PD pathophysiology and LID. OBJECTIVE: The objective of this study was to determine whether direct modulation of SNr GABAergic neurons and SNr projections to the pedunculopontine nucleus (PPN) regulates PD symptoms and LID in a mouse model. METHODS: We expressed Cre-recombinase activated channelrhodopsin-2 (ChR2) or halorhodopsin adeno-associated virus-2 (AAV2) vectors selectively in SNr GABAergic neurons of Vgat-IRES-Cre mice in a 6-hydroxydopamine model of PD to investigate whether direct optogenetic modulation of SNr neurons or their projections to the PPN regulates PD symptoms and LID expression. The forepaw stepping task, mouse LID rating scale, and open-field locomotion were used to assess akinesia and LID to test the effect of SNr modulation. RESULTS: Akinesia was improved by suppressing SNr neuron activity with halorhodopsin. LID was significantly reduced by increasing SNr neuronal activity with ChR2, which did not interfere with the antiakinetic effect of levodopa. Optical stimulation of ChR2 in SNr projections to the PPN recapitulated direct SNr stimulation. CONCLUSIONS: Modulation of SNr GABAergic neurons alters akinesia and LID expression in a manner consistent with the rate model of basal ganglia circuitry. Moreover, the projections from SNr to PPN likely mediate the antidyskinetic effect of increasing SNr neuronal activity, identifying a potential novel role for the PPN in LID. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Nitration of Hsp90 induces cell death.

Oxidative stress is a widely recognized cause of cell death associated with neurodegeneration, inflammation, and aging. Tyrosine nitration in these conditions has been reported extensively, but whether tyrosine nitration is a marker or plays a role in the cell-death processes was unknown. Here, we show that nitration of a single tyrosine residue on a small proportion of 90-kDa heat-shock protein (Hsp90), is sufficient to induce motor neuron death by the P2X7 receptor-dependent activation of the Fas pathway. Nitrotyrosine at position 33 or 56 stimulates a toxic gain of function that turns Hsp90 into a toxic protein. Using an antibody that recognizes the nitrated Hsp90, we found immunoreactivity in motor neurons of patients with amyotrophic lateral sclerosis, in an animal model of amyotrophic lateral sclerosis, and after experimental spinal cord injury. Our findings reveal that cell death can be triggered by nitration of a single protein and highlight nitrated Hsp90 as a potential target for the development of effective therapies for a large number of pathologies.

cAMP-responsive element-binding protein (CREB) and cAMP co-regulate activator protein 1 (AP1)-dependent regeneration-associated gene expression and neurite growth.

To regenerate damaged axons, neurons must express a cassette of regeneration-associated genes (RAGs) that increases intrinsic growth capacity and confers resistance to extrinsic inhibitory cues. Here we show that dibutyrl-cAMP or forskolin combined with constitutive-active CREB are superior to either agent alone in driving neurite growth on permissive and inhibitory substrates. Of the RAGs examined, only arginase 1 (Arg1) expression correlated with the increased neurite growth induced by the cAMP/CREB combination, both of which were AP1-dependent. This suggests that cAMP-induced AP1 activity is necessary and interacts with CREB to drive expression of RAGs relevant for regeneration and demonstrates that combining a small molecule (cAMP) with an activated transcription factor (CREB) stimulates the gene expression necessary to enhance axonal regeneration.

A screen for inducers of p21(waf1/cip1) identifies HIF prolyl hydroxylase inhibitors as neuroprotective agents with antitumor properties.

Preventing neuronal death is a priority for treating neurological diseases. However, therapies that inhibit pathological neuron loss could promote tumorigenesis by preventing the physiological death of cancerous cells. To avert this, we targeted the transcriptional upregulation of p21(waf1/cip1) (p21), an endogenous tumor suppressor with neuroprotective and pro-regenerative activity. We identified potential p21 indcuers by screening a FDA-approved drug and natural product small molecule library against hippocampal HT22 cells stably expressing a luciferase reporter driven by the proximal 60bp of the p21 promoter, and tested them for neuroprotection from glutathione depletion mediated oxidative stress, and cytotoxicity to cancer cell lines (DLD-1, Neuro-2A, SH-SY5Y, NGP, CHLA15, CHP212, and SK-N-SH) in vitro. Of the p21 inducers identified, only ciclopirox, a hypoxia-inducible factor prolyl-4-hydroxylase (HIF-PHD) inhibitor, simultaneously protected neurons from glutathione depletion and decreased cancer cell proliferation at concentrations that were not basally toxic to neurons. We found that other structurally distinct HIF-PHD inhibitors (desferrioxamine, 3,4-dihydroxybenzoate, and dimethyloxalyl glycine) also protected neurons at concentrations that killed cancer cells. HIF-PHD inhibitors stabilize HIF transcription factors, mediating genetic adaptation to hypoxia. While augmenting HIF stability is believed to promote tumorigenesis, we found that chronic HIF-PHD inhibition killed cancer cells, suggesting a protumorigenic role for these enzymes. Moreover, our findings suggest that PHD inhibitors can be used to treat neurological disease without significant concern for cell-autonomous tumor promotion.

In vitro ischemia suppresses hypoxic induction of hypoxia-inducible factor-1α by inhibition of synthesis and not enhanced degradation.

Hypoxia-inducible factor (HIF) mediates a broad, conserved adaptive response to hypoxia, and the HIF pathway is a potential therapeutic target in cerebral ischemia. This study investigated the mechanism by which in vitro ischemia (oxygen-glucose deprivation; OGD) affects canonical hypoxic HIF-1α stabilization. We validated the use of a reporter containing the oxygen-dependent degradation domain of HIF-1α fused to firefly luciferase (ODD-luc) to monitor quantitatively distinct biochemical events leading to hypoxic HIF-1α expression or stabilization in a human neuroblastoma cell line (SH-SY5Y). When OGD was imposed following a 2-hr hypoxic stabilization of ODD-luc, the levels of the reporter were reduced, consistent with prior models proposing that OGD enhances HIF prolylhydroxylase (PHD) activity. Surprisingly, PHD inhibitors and proteasome inhibitors do not stabilize ODD-luc in OGD. Furthermore, OGD does not affect the half-life of ODD-luc protein following hypoxia, suggesting that OGD abrogates hypoxic HIF-1α induction by reducing HIF-1α synthesis rather than by enhancing its degradation. We observed ATP depletion under OGD vs. hypoxia and propose that ATP depletion enhances translational suppression, overcoming the selective synthesis of HIF concurrent with global decreases in protein synthesis in hypoxia. Taken together, these findings biochemically characterize a practical reporter for monitoring HIF-1α levels and support a novel model for HIF regulation in an in vitro model of human ischemia.

Controlled enzymatic production of astrocytic hydrogen peroxide protects neurons from oxidative stress via an Nrf2-independent pathway.

Neurons rely on their metabolic coupling with astrocytes to combat oxidative stress. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) appears important for astrocyte-dependent neuroprotection from oxidative insults. Indeed, Nrf2 activators are effective in stroke, Parkinson disease, and Huntington disease models. However, key endogenous signals that initiate adaptive neuroprotective cascades in astrocytes, including activation of Nrf2-mediated gene expression, remain unclear. Hydrogen peroxide (H(2)O(2)) plays an important role in cell signaling and is an attractive candidate mediator of adaptive responses in astrocytes. Here we determine (i) the significance of H(2)O(2) in promoting astrocyte-dependent neuroprotection from oxidative stress, and (ii) the relevance of H(2)O(2) in inducing astrocytic Nrf2 activation. To control the duration and level of cytoplasmic H(2)O(2) production in astrocytes cocultured with neurons, we heterologously expressed the H(2)O(2)-producing enzyme Rhodotorula gracilis D-amino acid oxidase (rgDAAO) selectively in astrocytes. Exposure of rgDAAO-astrocytes to D-alanine lead to the concentration-dependent generation of H(2)O(2). Seven hours of low-level H(2)O(2) production (∼3.7 nmol·min·mg protein) in astrocytes protected neurons from oxidative stress, but higher levels (∼130 nmol·min·mg protein) were neurotoxic. Neuroprotection occurred without direct neuronal exposure to astrocyte-derived H(2)O(2), suggesting a mechanism specific to astrocytic intracellular signaling. Nrf2 activation mimicked the effect of astrocytic H(2)O(2) yet H(2)O(2)-induced protection was independent of Nrf2. Astrocytic protein tyrosine phosphatase inhibition also protected neurons from oxidative death, representing a plausible mechanism for H(2)O(2)-induced neuroprotection. These findings demonstrate the utility of rgDAAO for spatially and temporally controlling intracellular H(2)O(2) concentrations to uncover unique astrocyte-dependent neuroprotective mechanisms.

Neuron-derived extracellular vesicles to examine brain mTOR target engagement with sirolimus in patients with multiple system atrophy.

INTRODUCTION: Impaired autophagy is a pathogenic mechanism in the synucleinopathies. Sirolimus, a potent mTOR inhibitor and autophagy activator, had no beneficial effects in a randomized placebo-controlled trial in patients with multiple system atrophy (MSA). Whether sirolimus effectively inhibited brain mTOR activity was unknown. We aimed to evaluate if patients with MSA treated with sirolimus had evidence of inhibited brain mTOR pathways by measuring neuron-derived serum extracellular vesicles (NEVs). METHODS: Serum samples were collected from participants of the sirolimus-MSA trial, which randomized patients to sirolimus (2-6 mg/day) or placebo for 48 weeks. NEVs were immunoprecipitated with three antibodies-against neurons. Brain mTOR engagement was quantified as the change in the NEV phosphorylated mTOR (p-mTOR) to total-mTOR (tot-mTOR) ratio after 48 weeks of sirolimus. RESULTS: Samples from 27 patients [mean (±SD) age, 59.2±7 years, 15 (55.5%) men] were analyzed (19 sirolimus, 8 placebo). Treated- and placebo-patients had similar p-mTOR:tot-mTOR ratio at 24 (placebo: 0.248 ± 0.03, sirolimus: 0.289 ± 0.02; P = 0.305) and 48 weeks (placebo: 0.299 ± 0.05, sirolimus: 0.261 ± 0.03; P = 0.544). The tot-mTOR, p-mTOR, or their ratio levels were not associated with Unified MSA Rating Scale (UMSARS) worsening. DISCUSSION: These results are consistent with no brain mTOR engagement by oral sirolimus up to 6 mg/day. NEV-based biomarkers are a rational approach to investigating target engagement in clinical trials of brain-targeted therapeutics.

Defining the molecular identity and morphology of glia limitans superficialis astrocytes in mouse and human.

Astrocytes are a highly abundant glial cell type that perform critical homeostatic functions in the central nervous system. Like neurons, astrocytes have many discrete heterogenous subtypes. The subtype identity and functions are, at least in part, associated with their anatomical location and can be highly restricted to strategically important anatomical domains. Here, we report that astrocytes forming the glia limitans superficialis, the outermost border of brain and spinal cord, are a highly specialized astrocyte subtype and can be identified by a single marker: Myocilin (Myoc). We show that Myoc+ astrocytes cover the entire brain and spinal cord surface, exhibit an atypical morphology, and are evolutionarily conserved from rodents to humans. Identification of this highly specialized astrocyte subtype will advance our understanding of CNS homeostasis and potentially be targeted for therapeutic intervention to combat peripheral inflammatory effects on the CNS.

A large-scale chemical screen for regulators of the arginase 1 promoter identifies the soy isoflavone daidzeinas a clinically approved small molecule that can promote neuronal protection or regeneration via a cAMP-independent pathway.

An ideal therapeutic for stroke or spinal cord injury should promote survival and regeneration in the CNS. Arginase 1 (Arg1) has been shown to protect motor neurons from trophic factor deprivation and allow sensory neurons to overcome neurite outgrowth inhibition by myelin proteins. To identify small molecules that capture Arg1's protective and regenerative properties, we screened a hippocampal cell line stably expressing the proximal promoter region of the arginase 1 gene fused to a reporter gene against a library of compounds containing clinically approved drugs. This screen identified daidzein as a transcriptional inducer of Arg1. Both CNS and PNS neurons primed in vitro with daidzein overcame neurite outgrowth inhibition from myelin-associated glycoprotein, which was mirrored by acutely dissociated and cultured sensory neurons primed in vivo by intrathecal or subcutaneous daidzein infusion. Further, daidzein was effective in promoting axonal regeneration in vivo in an optic nerve crush model when given intraocularly without lens damage, or most importantly, when given subcutaneously after injury. Mechanistically, daidzein requires transcription and induction of Arg1 activity for its ability to overcome myelin inhibition. In contrast to canonical Arg1 activators, daidzein increases Arg1 without increasing CREB phosphorylation, suggesting its effects are cAMP-independent. Accordingly, it may circumvent known CNS side effects of some cAMP modulators. Indeed, daidzein appears to be safe as it has been widely consumed in soy products, crosses the blood-brain barrier, and is effective without pretreatment, making it an ideal candidate for development as a therapeutic for spinal cord injury or stroke.

A rapid α-synuclein seed assay of Parkinson's disease CSF panel shows high diagnostic accuracy.

BACKGROUND: Assays that specifically measure α-synuclein seeding activity in biological fluids could revolutionize the diagnosis of Parkinson's disease. Recent improvements in α-synuclein real-time quaking-induced conversion assays of cerebrospinal fluid have dramatically reduced reaction times from 5-13 days down to 1-2 days. OBJECTIVE: To test our improved assay against a panel of cerebrospinal fluid specimens from patients with Parkinson's disease and healthy controls from the MJ Fox Foundation/NINDS BioFIND collection. METHODS: Specimens collected from healthy controls and patients with clinically typical moderate-to-advanced Parkinson's disease were tested without prior knowledge of disease status. Correlative analyses between assay parameters and clinical measures were performed by an independent investigator. RESULTS: BioFIND samples gave positive signals in 105/108 (97%) Parkinson's disease cases versus 11/85 (13%) healthy controls. Receiver operating characteristic analyses of diagnosis of cases versus healthy controls gave areas under the curve of 95%. Beyond binary positive/negative determinations, only weak correlations were observed between various assay response parameters and Parkinson's disease clinical measures or other cerebrospinal fluid analytes. Of note, REM sleep behavioral disorder questionnaire scores correlated with the reaction times needed to reach 50% maximum fluorescence. Maximum fluorescence was inversely correlated with Unified Parkinson's Disease Rating Scale motor scores, which was driven by the patients without REM sleep behavioral disorder. CONCLUSIONS: Our improved α-synuclein seed amplification assay dramatically reduces the time needed to diagnose Parkinson's disease while maintaining the high-performance standards associated with previous α-synuclein seed assays, supporting the clinical utility of this assay for Parkinson's disease diagnosis.

Failure to Upregulate the RNA Binding Protein ZBP After Injury Leads to Impaired Regeneration in a Rodent Model of Diabetic Peripheral Neuropathy.

Most diabetes patients eventually suffer from peripheral nerve degeneration. Unfortunately, there is no treatment for the condition and its mechanisms are not well understood. There is, however, an emerging consensus that the inability of peripheral nerves to regenerate normally after injury contributes to the pathophysiology. We have previously shown that regeneration of peripheral axons requires local axonal translation of a pool of axonal mRNAs and that the levels and members of this axonal mRNA pool are altered in response to injury. Here, we show that following sciatic nerve injury in a streptozotocin rodent model of type I diabetes, this mobilization of RNAs into the injured axons is attenuated and correlates with decreased axonal regeneration. This failure of axonal RNA localization results from decreased levels of the RNA binding protein ZBP1. Over-expression of ZBP1 rescues the in vitro growth defect in injured dorsal root ganglion neurons from diabetic rodents. These results provide evidence that decreased neuronal responsiveness to injury in diabetes is due to a decreased ability to alter the pool of axonal mRNAs available for local translation, and may open new therapeutic opportunities for diabetic peripheral neuropathy.

Alterations in the intrinsic properties of striatal cholinergic interneurons after dopamine lesion and chronic L-DOPA.

Changes in striatal cholinergic interneuron (ChI) activity are thought to contribute to Parkinson's disease pathophysiology and dyskinesia from chronic L-3,4-dihydroxyphenylalanine (L-DOPA) treatment, but the physiological basis of these changes is unknown. We find that dopamine lesion decreases the spontaneous firing rate of ChIs, whereas chronic treatment with L-DOPA of lesioned mice increases baseline ChI firing rates to levels beyond normal activity. The effect of dopamine loss on ChIs was due to decreased currents of both hyperpolarization-activated cyclic nucleotide-gated (HCN) and small conductance calcium-activated potassium (SK) channels. L-DOPA reinstatement of dopamine normalized HCN activity, but SK current remained depressed. Pharmacological blockade of HCN and SK activities mimicked changes in firing, confirming that these channels are responsible for the molecular adaptation of ChIs to dopamine loss and chronic L-DOPA treatment. These findings suggest that targeting ChIs with channel-specific modulators may provide therapeutic approaches for alleviating L-DOPA-induced dyskinesia in PD patients.

An Assessment of LRRK2 Serine 935 Phosphorylation in Human Peripheral Blood Mononuclear Cells in Idiopathic Parkinson's Disease and G2019S LRRK2 Cohorts.

The phosphorylated form of LRRK2, pS935 LRRK2, has been proposed as a target modulation biomarker for LRRK2 inhibitors. The primary aim of the study was to characterize and qualify this biomarker for therapeutic trials of LRRK2 inhibitors in Parkinson's disease (PD). To this end, analytically validated assays were used to monitor levels of pS935 LRRK2 and total LRRK2 in peripheral blood mononuclear cells (PBMCs) from the following donor groups: healthy controls, idiopathic PD, and G2019S carriers with and without PD. Neither analyte correlated with age, gender, or disease severity. While total LRRK2 levels were similar across the four groups, there was a significant reduction in pS935 LRRK2 levels in disease-manifesting G2019S carriers compared to idiopathic PD. In aggregate, these data indicate that phosphorylation of LRRK2 at S935 may reflect a state marker for G2019S LRRK2-driven PD, the underlying biology for which requires investigation in future studies. This study also provides critical foundational data to inform the integration of pS935 and total LRRK2 levels as biomarkers in therapeutic trials of LRRK2 kinase inhibitors.

Metformin therapy in a transgenic mouse model of Huntington's disease.

Huntington's disease (HD) is a hereditary neurodegenerative disease that leads to striatal degeneration and a severe movement disorder. We used a transgenic mouse model of HD (the R6/2 line with approximately 150 glutamine repeats) to test a new therapy for this disease. We treated HD mice with metformin, a widely used anti-diabetes drug, in the drinking water (0, 2 or 5mg/ml) starting at 5 weeks of age. Metformin treatment significantly prolonged the survival time of male HD mice at the 2mg/ml dose (20.1% increase in lifespan) without affecting fasting blood glucose levels. This dose of metformin also decreased hind limb clasping time in 11-week-old mice. The higher dose did not prolong survival, and neither dose of metformin was effective in female HD mice. Collectively, our results suggest that metformin may be worth further investigation in additional HD models.

A sensitive and selective assay of neuronal degeneration in cell culture.

We have developed a simple and sensitive assay to quantify neuron-specific death in primary cell cultures that represents a significant improvement over more commonly used methods including manual cell counting and lactate dehydrogenase release. This new method selectively detects neuronal death by combining immunolabeling for a neuron-specific marker with the ease, sensitivity, and speed of an enzyme-linked fluorescence assay. Using microtubule associated protein 2 (MAP2) as a neuron-specific marker, we assessed glutamate-receptor mediated neurotoxicity in neuron-enriched cultures and in mixed neuronal/glial cultures established from mouse forebrain and compared these results to neuronal death measured by lactate dehydrogenase (LDH) release. We were able to achieve statistically significant differences in toxicity between intermediately toxic concentrations of glutamate (30, 50, and 100 microM) with the MAP2 assay, while we were not able to discriminate among these concentrations with the LDH assay. We were also able to measure hydrogen peroxide-induced neuronal death, and demonstrate neuroprotection by antioxidant addition. This new assay is easily adaptable to high-throughput in vitro screens of neurodegeneration and of neuroprotective therapies.

What makes a RAG regeneration associated?

Regenerative failure remains a significant barrier for functional recovery after central nervous system (CNS) injury. As such, understanding the physiological processes that regulate axon regeneration is a central focus of regenerative medicine. Studying the gene transcription responses to axon injury of regeneration competent neurons, such as those of the peripheral nervous system (PNS), has provided insight into the genes associated with regeneration. Though several individual "regeneration-associated genes" (RAGs) have been identified from these studies, the response to injury likely regulates the expression of functionally coordinated and complementary gene groups. For instance, successful regeneration would require the induction of genes that drive the intrinsic growth capacity of neurons, while simultaneously downregulating the genes that convey environmental inhibitory cues. Thus, this view emphasizes the transcriptional regulation of gene "programs" that contribute to the overall goal of axonal regeneration. Here, we review the known RAGs, focusing on how their transcriptional regulation can reveal the underlying gene programs that drive a regenerative phenotype. Finally, we will discuss paradigms under which we can determine whether these genes are injury-associated, or indeed necessary for regeneration.

HIF prolyl hydroxylase inhibitors prevent neuronal death induced by mitochondrial toxins: therapeutic implications for Huntington's disease and Alzheimer's disease.

Mitochondrial dysfunction is a central feature of a number of acute and chronic neurodegenerative conditions, but clinically approved therapeutic interventions are only just emerging. Here we demonstrate the potential clinical utility of low molecular weight inhibitors of the hypoxia inducible factor prolyl-4-hydroxylases (HIF PHDs) in preventing mitochondrial toxin-induced cell death in mouse striatal neurons that express a "knock-in" mutant Huntingtin allele. Protection from 3-nitropropionic acid (3-NP, a complex II inhibitor)-induced toxicity by HIF PHD inhibition occurs without rescue of succinate dehydrogenase activity. Although HIF-1alpha mRNA is dramatically induced by mutant huntingtin, HIF-1alpha depletion by short interfering RNAs (siRNA) does not affect steady-state viability or protection from 3-NP-induced death by HIF PHD inhibitors in these cells. Moreover, 3-NP-induced complex II inhibition in control or mutant striatal neurons does not lead to activation of HIF-dependent transcription. HIF PHD inhibition also protects cortical neurons from 3-NP-induced cytotoxicity. Protection of cortical neurons by HIF PHD inhibition correlates with enhanced VEGF but not PGC-1alpha gene expression. Together, these findings suggest that HIF PHD inhibitors are promising candidates for preventing cell death in conditions such as Huntington's disease and Alzheimer's disease that are associated with metabolic stress in the central nervous system.

Bioenergetic and oxidative effects of free 3-nitrotyrosine in culture: selective vulnerability of dopaminergic neurons and increased sensitivity of non-dopaminergic neurons to dopamine oxidation.

Protein bound and free 3-nitrotyrosine (3NT) levels are elevated in neurodegenerative diseases and have been used as evidence for peroxynitrite generation. Intrastriatal injection of free 3NT causes dopaminergic neuron injury and represents a new mouse model of Parkinson's disease (PD). We are investigating the nature of free 3NT neurotoxicity. In primary ventral midbrain cultures, free 3NT damaged dopaminergic neurons, while adjacent non-dopaminergic neurons were unaffected. Combined treatment with free 3NT and subtoxic amounts of dopamine caused extensive death of non-dopaminergic forebrain neurons in culture. Free 3NT alone directly inhibited mitochondrial complex I, decreased ATP, sensitized neurons to mitochondrial depolarization, and increased superoxide production. Subtoxic concentrations of rotenone (instead of free 3NT) caused similar results. Additionally, free 3NT and dopamine combined increased extraneuronal hydrogen peroxide and decreased intraneuronal glutathione levels more than dopamine alone. Oxidative and bioenergetic processes have been proposed to contribute to neurodegeneration in PD. As free 3NT is a compound that is increased in PD, damages dopamine neurons in vivo and in vitro and has detrimental effects on neuronal bioenergetics, it is possible that free 3NT is an endogenous contributing factor to neuronal loss, in addition to being a marker of oxidative and nitrative processes.