Barry J. Hoffer’s research while affiliated with Cleveland University and other places

Background Traumatic Brain Injury (TBI) is a major risk factor for neurodegenerative disorders such as Parkinson’s disease (PD) and Alzheimer’s disease (AD), with neuroinflammation playing a critical role in the secondary cell death that exacerbates the initial injury. While targeting neuroinflammation holds significant therapeutic promise, clinical trials of available anti-inflammatory agents have fallen short. 3-Mono-thiopomalidomide (3-MP), a novel immunomodulatory imide drug (IMiD), was designed to curb inflammation without the adverse effects of traditional IMiDs and was evaluated across models involving neuroinflammation. Methods 3-MP anti-inflammatory activity was evaluated across cellular (RAW 264.7, IMG cells) and mouse studies following lipopolysaccharide (LPS)-challenge (for pro- and anti-inflammatory cytokines/chemokines), and mice subjected to controlled cortical impact (CCI) moderate traumatic brain injury (TBI). 3-MP human cereblon binding, including neosubstrate and molecular modeling evaluation, as well as chicken teratogenicity, ex vivo mouse and human stability studies, and mouse pharmacokinetics were appraised. Results 3-MP binds human cereblon, a key protein in the E3 ubiquitin ligase complex, without triggering downstream cascades leading to thalidomide-like teratogenicity in chicken embryos. 3-MP reduces pro-inflammatory markers in LPS-stimulated mouse macrophage and microglial cell cultures, and lowers pro-inflammatory cytokine/chemokine levels in plasma and brain of mice challenged with systemic LPS without lowering anti-inflammatory IL-10. 3-MP readily enters brain following systemic administration, and achieves a brain/plasma concentration ratio of 0.44–0.47. 3-MP mitigates behavioral impairments and reduces activation of astrocytes and microglia in mice challenged with CCI TBI. Conclusion 3-MP represents a promising new class of thalidomide-like IMiDs with potent anti-inflammatory effects that offers potential for treating TBI and possibly other neurodegenerative diseases possessing a prominent neuroinflammatory component.

An overactive neuroinflammatory response is often evident in the elderly and is a significant contributor to brain tissue damage following acute ischemic stroke. Such an inflammatory response is largely mediated by microglial cells and peripheral blood mononuclear cells (PBMCs). Classical anti-inflammatory agents have not proved clinically effective in mitigating the impact of ischemic stroke but have highlighted targets for new drug development, in particular excessive proinflammatory cytokine release. The immunomodulatory imide drug (IMiD) class has shown potential in reducing neuroinflammation and switching microglial phenotypic expression away from a proinflammatory to a regenerative anti-inflammatory one. 3-Monothiopomalidomide (3-MP), a new IMiD, has a brain/plasma concentration ratio of 0.5 to 0.6, an oral bioavailability of 38.5%, and a monophasic disappearance of half-life 3.2 h following oral administration. 3-MP pretreatment mitigates lipopolysaccharide (LPS)-induced inflammation in cellular human PBMCs and, in rat studies, 3-MP pretreatment lowers proinflammatory cytokine levels in the conditioned media and in plasma and the brain, respectively. Administered systemically to rats challenged with middle cerebral artery occlusion (MCAo) and reperfusion, 3-MP post-MCAo treatment reduced infarction volume; improved body asymmetry, a behavioral measure of stroke impact; and lowered inflammation. In summary, 3-MP exerted neuroprotective effects via anti-inflammatory actions against MCAo-induced ischemic injury and represents a therapeutic that warrants further investigation as a treatment for brain damage and related disorders associated with excessive inflammation.

Background Epidemiological studies report an elevated risk of neurodegenerative disorders, particularly Parkinson’s disease (PD), in patients with type 2 diabetes mellitus (T2DM) that is mitigated in those prescribed incretin mimetics or dipeptidyl peptidase 4 inhibitors (DPP‐4Is). Incretin mimetic repurposing appears promising in human PD and Alzheimer’s disease (AD) clinical trials. DPP‐4Is are yet to be evaluated in PD or AD human studies. Methods Incretin mimetics have been evaluated in multiple cellular/animal PD models, including in 6‐hdroxydopamine (6‐OHDA) rats, and have demonstrated efficacy. The clinically approved DPP‐4Is, sitagliptin (Januvia) and PF‐00734200 (gosogliptin/Saterex) were hence evaluated in the classic 6‐OHDA unilateral medial forebrain bundle rat PD model to evaluate their repurposing potential when administered as human equivalent oral doses. Equivalent doses then were administered to naive nonhuman primates (NHPs) to evaluate whether biomarkers of efficacy in rat could be reproduced in NHPs as a further translational step towards human studies. Pharmacokinetics, DPP‐4 inhibition, GLP‐1/GIP and dopamine levels, together with dopaminergic and neuroinflammatory markers, and GLP‐1/GIP receptor levels were quantified. Results Sitagliptin/PF‐00734,200 pre‐ or post‐treatment mitigated 6‐OHDA‐induced dopaminergic neurodegeneration, dopamine level loss and neuroinflammation, and augmented neurogenesis in lesioned substantia nigra pars compacta and in striatum, and reduced classical methamphetamine‐induced rotation in rats. This efficacy associated with 70‐80% plasma and 20‐30% brain DPP‐4 inhibition, and with elevated plasma and brain GLP‐1/GIP levels. Alike plasma/CSF DPP‐4 inhibition and elevated GLP‐1/GIP levels were determined in NHPs administered rat equivalent (human translational) sitagliptin doses. In relation to the drug targets, brain GLP‐1/GIP receptor protein levels were age‐dependently maintained in rodents, preserved in rats challenged with 6‐OHDA, and in humans with PD. Combined GLP‐1+GIP receptor activation in neuronal cultures resulted in neurotrophic/neuroprotective actions superior to single agonists alone – particularly when combined with a DPP‐4I Conclusions These studies support further evaluation of repurposing clinically approved drugs that elevate plasma/brain/CSF GLP‐1/GIP as a treatment strategy for neurodegenerative disorders. Incretin mimetics are already in clinical evaluation in PD and AD. Similarly, the repurposing of gliptins warrants evaluation both alone and in combination with an effective incretin mimetic.

Background Traumatic brain injury (TBI) causes axon tearing and synapse degradation, resulting in multiple neurological dysfunctions and exacerbation of early neurodegeneration; the repair of axonal and synaptic structures is critical for restoring neuronal function. C-C Motif Chemokine Ligand 5 (CCL5) shows many neuroprotective activities. Method A close-head weight-drop system was used to induce mild brain trauma in C57BL/6 (wild-type, WT) and CCL5 knockout (CCL5-KO) mice. The mNSS score, rotarod, beam walking, and sticker removal tests were used to assay neurological function after mTBI in different groups of mice. The restoration of motor and sensory functions was impaired in CCL5-KO mice after one month of injury, with swelling of axons and synapses from Golgi staining and reduced synaptic proteins-synaptophysin and PSD95. Administration of recombinant CCL5 (Pre-treatment: 300 pg/g once before injury; or post-treatment: 30 pg/g every 2 days, since 3 days after injury for 1 month) through intranasal delivery into mouse brain improved the motor and sensory neurological dysfunctions in CCL5-KO TBI mice. Results Proteomic analysis using LC-MS/MS identified that the “Nervous system development and function”-related proteins, including axonogenesis, synaptogenesis, and myelination signaling pathways, were reduced in injured cortex of CCL5-KO mice; both pre-treatment and post-treatment with CCL5 augmented those pathways. Immunostaining and western blot analysis confirmed axonogenesis and synaptogenesis related Semaphorin, Ephrin, p70S6/mTOR signaling, and myelination-related Neuregulin/ErbB and FGF/FAK signaling pathways were up-regulated in the cortical tissue by CCL5 after brain injury. We also noticed cortex redevelopment after long-term administration of CCL5 after brain injury with increased Reelin positive Cajal-Rerzius Cells and CXCR4 expression. CCL5 enhanced the growth of cone filopodia in a primary neuron culture system; blocking CCL5’s receptor CCR5 by Maraviroc reduced the intensity of filopodia in growth cone and also CCL5 mediated mTOR and Rho signalling activation. Inhibiting mTOR and Rho signaling abolished CCL5 induced growth cone formation. Conclusions CCL5 plays a critical role in starting the intrinsic neuronal regeneration system following TBI, which includes growth cone formation, axonogenesis and synaptogensis, remyelination, and the subsequent proper wiring of cortical circuits. Our study underscores the potential of CCL5 as a robust therapeutic stratagem in treating axonal injury and degeneration during the chronic phase after mild brain injury. Graphical Abstract

Background The serotonin (5-HT) system can manipulate the processing of exogenous L-DOPA in the DA-denervated striatum, resulting in the modulation of L-DOPA-induced dyskinesia (LID). Objective To characterize the effects of the serotonin precursor 5-hydroxy-tryptophan (5-HTP) or the serotonin transporter (SERT) inhibitor, Citalopram on L-DOPA-induced behavior, neurochemical signals, and underlying protein expressions in an animal model of Parkinson’s disease. Methods MitoPark (MP) mice at 20 weeks of age, subjected to a 14-day administration of L-DOPA/Carbidopa, displayed dyskinesia, referred to as LID. Subsequent investigations explored the effects of 5-HT-modifying agents, such as 5-HTP and Citalopram, on abnormal involuntary movements (AIMs), locomotor activity, neurochemical signals, serotonin transporter activity, and protein expression in the DA-denervated striatum of LID MP mice. Results 5-HTP exhibited duration-dependent suppressive effects on developing and established LID, especially related to abnormal limb movements observed in L-DOPA-primed MP mice. However, Citalopram, predominantly suppressed abnormal axial movement induced by L-DOPA in LID MP mice. We demonstrated that 5-HTP could decrease L-DOPA-upregulation of DA turnover rates while concurrently upregulating 5-HT metabolism. Additionally, 5-HTP was shown to reduce the expressions of p-ERK and p-DARPP-32 in the striatum of LID MP mice. The effect of Citalopram in alleviating LID development may be attributed to downregulation of SERT activity in the dorsal striatum of LID MP mice. Conclusions While both single injection of 5-HTP and Citalopram effectively mitigated the development of LID, the difference in mitigation of AIM subtypes may be linked to the unique effects of these two serotonergic agents on L-DOPA-derived DA and 5-HT metabolism.

Cholinergic innervation in brain is involved in modulating neurovascular function including cerebral blood flow hemodynamics in response to neuronal activity. Cholinergic deficit is associated with pathophysiology in Alzheimer’s disease, albeit the etiology remains to clarify. In current study, neocortex cerebral blood flow response to acetylcholine was evaluated by Laser-Doppler Flowmetry in 3xTgAD Alzheimer’s disease model) and wild type mice of two age groups. The peak of cerebral blood flow to acetylcholine (i.v.) from baseline levels (% ΔrCBF) were higher in young 3xTgAD vs. in wild type mice (48.35; CI:27.03-69.67 vs. 22.70; CI:15.5-29.91, p < 0.05); this was reversed in old 3xTgAD mice (21.44; CI:2.52-40.35 vs. 23.25; CI:23.25-39). Choline acetyltransferase protein was reduced in neocortex, while cerebrovascular reactivity to acetylcholine was preserved in young 3xTgAD mice. This suggests of endogenous acetylcholine deficit and possible cholinergic denervation from selected cholinergic nuclei within basal forebrain. The early deposition of tauopathy moieties (mutant hTau and pTau181) and its coincidence in cholinergic cell clusters, were observed occasionally at basal forebrain of 3xTgAD mice including substantia innominate, nucleus Basalis of Meynert and nucleus of horizontal limb diagonal band of Broca. A prominent feature was microglia interacting tauopathy and demonstrated a variety of morphology changes particularly when located in proximity to tauopathy. The microglia ramified phenotype was reduced as evaluated by the ramification index and Fractal analysis. Increased microglia senescence, identified as SASP (senescence-associated secretory phenotype), was colocalization with p16Ink4ɑ, a marker of irreversible cell cycle arrest in old 3xTgAD vs. wild type mice (p = 0.001). The p16Ink4ɑ was also observed in neuronal cells bearing tauopathy within basal forebrain of 3xTgAD mice. TNF-ɑ, the pro-inflammatory cytokine elevated persistently in microglia (Peason’s correlation coefficient = 0.62), and the loss of cholinergic cells in vulnerable basal forebrain environment was indicated by image analysis in 3xTgAD mice, which linked to the cholinergic deficits in neocortex rCBF hemodynamics. Our study revealed the early change of CBF hemodynamics to acetylcholine in 3xTgAD model. As a major effector of brain innate immune activation, microglia SASP with age related disease progression is indicative of immune cell senescence, which contributes to chronic inflammation and cholinergic deficits at basal forebrain. Targeting neuroinflammation and senescence may mitigate cholinergic pathophysiology in Alzheimer’s disease.

Background Mitochondria are essential organelles involved in cellular energy production. Changes in mitochondrial function can lead to dysfunction and cell death in aging and age-related disorders. Recent research suggests that mitochondrial dysfunction is closely linked to neurodegenerative diseases. Glucagon-like peptide-1 receptor (GLP-1R) agonist has gained interest as a potential treatment for Parkinson's disease (PD). However, the exact mechanisms responsible for the therapeutic effects of GLP-1R-related agonists are not yet fully understood. Methods In this study, we explores the effects of early treatment with PT320, a sustained release formulation of the GLP-1R agonist Exenatide, on mitochondrial functions and morphology in a progressive PD mouse model, the MitoPark (MP) mouse. Results Our findings demonstrate that administration of a clinically translatable dose of PT320 ameliorates the reduction in tyrosine hydroxylase expression, lowers reactive oxygen species (ROS) levels, and inhibits mitochondrial cytochrome c release during nigrostriatal dopaminergic denervation in MP mice. PT320 treatment significantly preserved mitochondrial function and morphology but did not influence the reduction in mitochondria numbers during PD progression in MP mice. Genetic analysis indicated that the cytoprotective effect of PT320 is attributed to a reduction in the expression of mitochondrial fission protein 1 (Fis1) and an increase in the expression of optic atrophy type 1 (Opa1), which is known to play a role in maintaining mitochondrial homeostasis and decreasing cytochrome c release through remodeling of the cristae. Conclusion Our findings suggest that the early administration of PT320 shows potential as a neuroprotective treatment for PD, as it can preserve mitochondrial function. Through enhancing mitochondrial health by regulating Opa1 and Fis1, PT320 presents a new neuroprotective therapy in PD. Graphical Abstract

Epidemiological studies report an elevated risk of Parkinson's disease (PD) in patients with type 2 diabetes mellitus (T2DM) that is mitigated in those prescribed dipeptidyl peptidase 4 (DPP-4) inhibitors. With an objective to characterize clinically translatable doses of DPP-4 inhibitors (gliptins) in a well-characterized PD rodent model, sitagliptin, PF-00734,200 or vehicle were orally administered to rats initiated either 7-days before or 7-days after unilateral medial forebrain bundle 6-hydroxydopamine (6-OHDA) lesioning. Measures of dopaminergic cell viability, dopamine content, neuroinflammation and neurogenesis were evaluated thereafter in ipsi- and contralateral brain. Plasma and brain incretin and DPP-4 activity levels were quantified. Furthermore, brain incretin receptor levels were age-dependently evaluated in rodents, in 6-OHDA challenged animals and human subjects with/without PD. Cellular studies evaluated neurotrophic/neuroprotective actions of combined incretin administration. Pre-treatment with oral sitagliptin or PF-00734,200 reduced methamphetamine (meth)-induced rotation post-lesioning and dopaminergic degeneration in lesioned substantia nigra pars compacta (SNc) and striatum. Direct intracerebroventricular gliptin administration lacked neuroprotective actions, indicating that systemic incretin-mediated mechanisms underpin gliptin-induced favorable brain effects. Post-treatment with a threefold higher oral gliptin dose, likewise, mitigated meth-induced rotation, dopaminergic neurodegeneration and neuroinflammation, and augmented neurogenesis. These gliptin-induced actions associated with 70–80% plasma and 20–30% brain DPP-4 inhibition, and elevated plasma and brain incretin levels. Brain incretin receptor protein levels were age-dependently maintained in rodents, preserved in rats challenged with 6-OHDA, and in humans with PD. Combined GLP-1 and GIP receptor activation in neuronal cultures resulted in neurotrophic/neuroprotective actions superior to single agonists alone. In conclusion, these studies support further evaluation of the repurposing of clinically approved gliptins as a treatment strategy for PD. Supplementary Information The online version contains supplementary material available at 10.1007/s11357-024-01116-0.

The endogenous incretins glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) possess neurotrophic, neuroprotective, and anti-neuroinflammatory actions. The dipeptidyl peptidase 4 (DPP-4) inhibitor sitagliptin reduces degradation of endogenous GLP-1 and GIP, and, thereby, extends the circulation of these protective peptides. The current nonhuman primate (NHP) study evaluates whether human translational sitagliptin doses can elevate systemic and central nervous system (CNS) levels of GLP-1/GIP in naive, non-lesioned NHPs, in line with our prior rodent studies that demonstrated sitagliptin efficacy in preclinical models of Parkinson’s disease (PD). PD is an age-associated neurodegenerative disorder whose current treatment is inadequate. Repositioning of the well-tolerated and efficacious diabetes drug sitagliptin provides a rapid approach to add to the therapeutic armamentarium for PD. The pharmacokinetics and pharmacodynamics of 3 oral sitagliptin doses (5, 20, and 100 mg/kg), equivalent to the routine clinical dose, a tolerated higher clinical dose and a maximal dose in monkey, were evaluated. Peak plasma sitagliptin levels were aligned both with prior reports in humans administered equivalent doses and with those in rodents demonstrating reduction of PD associated neurodegeneration. Although CNS uptake of sitagliptin was low (cerebrospinal fluid (CSF)/plasma ratio 0.01), both plasma and CSF concentrations of GLP-1/GIP were elevated in line with efficacy in prior rodent PD studies. Additional cellular studies evaluating human SH-SY5Y and primary rat ventral mesencephalic cultures challenged with 6-hydroxydopamine, established cellular models of PD, demonstrated that joint treatment with GLP-1 + GIP mitigated cell death, particularly when combined with DPP-4 inhibition to maintain incretin levels. In conclusion, this study provides a supportive translational step towards the clinical evaluation of sitagliptin in PD and other neurodegenerative disorders for which aging, similarly, is the greatest risk factor.