Fosgonimeton attenuates amyloid-beta toxicity in preclinical models of Alzheimer's disease.

Positive modulation of hepatocyte growth factor (HGF) signaling may represent a promising therapeutic strategy for Alzheimer's disease (AD) based on its multimodal neurotrophic, neuroprotective, and anti-inflammatory effects addressing the complex pathophysiology of neurodegeneration. Fosgonimeton is a small-molecule positive modulator of the HGF system that has demonstrated neurotrophic and pro-cognitive effects in preclinical models of dementia. Herein, we evaluate the neuroprotective potential of fosgonimeton, or its active metabolite, fosgo-AM, in amyloid-beta (Aß)-driven preclinical models of AD, providing mechanistic insight into its mode of action. In primary rat cortical neurons challenged with Aß (Aß1-42), fosgo-AM treatment significantly improved neuronal survival, protected neurite networks, and reduced tau hyperphosphorylation. Interrogation of intracellular events indicated that cortical neurons treated with fosgo-AM exhibited a significant decrease in mitochondrial oxidative stress and cytochrome c release. Following Aß injury, fosgo-AM significantly enhanced activation of pro-survival effectors ERK and AKT, and reduced activity of GSK3ß, one of the main kinases involved in tau hyperphosphorylation. Fosgo-AM also mitigated Aß-induced deficits in Unc-like kinase 1 (ULK1) and Beclin-1, suggesting a potential effect on autophagy. Treatment with fosgo-AM protected cortical neurons from glutamate excitotoxicity, and such effects were abolished in the presence of an AKT or MEK/ERK inhibitor. In vivo, fosgonimeton administration led to functional improvement in an intracerebroventricular Aß25-35 rat model of AD, as it significantly rescued cognitive function in the passive avoidance test. Together, our data demonstrate the ability of fosgonimeton to counteract mechanisms of Aß-induced toxicity. Fosgonimeton is currently in clinical trials for mild-to-moderate AD (NCT04488419; NCT04886063).

ATH-1105, a small-molecule positive modulator of the neurotrophic HGF system, is neuroprotective, preserves neuromotor function, and extends survival in preclinical models of ALS.

Introduction: Amyotrophic lateral sclerosis (ALS), a progressive and fatal neurodegenerative disorder, primarily affects the motor neurons of the brain and spinal cord. Like other neurodegenerative conditions, ongoing pathological processes such as increased inflammation, excitotoxicity, and protein accumulation contribute to neuronal death. Hepatocyte growth factor (HGF) signaling through the MET receptor promotes pro-survival, anti-apoptotic, and anti-inflammatory effects in multiple cell types, including the neurons and support cells of the nervous system. This pleiotropic system is therefore a potential therapeutic target for treatment of neurodegenerative disorders such as ALS. Here, we test the effects of ATH-1105, a small-molecule positive modulator of the HGF signaling system, in preclinical models of ALS. Methods: In vitro, the impact of ATH-1105 on HGF-mediated signaling was assessed via phosphorylation assays for MET, extracellular signal-regulated kinase (ERK), and protein kinase B (AKT). Neuroprotective effects of ATH-1105 were evaluated in rat primary neuron models including spinal motor neurons, motor neuron-astrocyte cocultures, and motor neuron-human muscle cocultures. The anti-inflammatory effects of ATH-1105 were evaluated in microglia- and macrophage-like cell systems exposed to lipopolysaccharide (LPS). In vivo, the impact of daily oral treatment with ATH-1105 was evaluated in Prp-TDP43A315T hemizygous transgenic ALS mice. Results: In vitro, ATH-1105 augmented phosphorylation of MET, ERK, and AKT. ATH-1105 attenuated glutamate-mediated excitotoxicity in primary motor neurons and motor neuron- astrocyte cocultures, and had protective effects on motor neurons and neuromuscular junctions in motor neuron-muscle cocultures. ATH-1105 mitigated LPS-induced inflammation in microglia- and macrophage-like cell systems. In vivo, ATH-1105 treatment resulted in improved motor and nerve function, sciatic nerve axon and myelin integrity, and survival in ALS mice. Treatment with ATH-1105 also led to reductions in levels of plasma biomarkers of inflammation and neurodegeneration, along with decreased pathological protein accumulation (phospho-TDP-43) in the sciatic nerve. Additionally, both early intervention (treatment initiation at 1 month of age) and delayed intervention (treatment initiation at 2 months of age) with ATH-1105 produced benefits in this preclinical model of ALS. Discussion: The consistent neuroprotective and anti-inflammatory effects demonstrated by ATH-1105 preclinically provide a compelling rationale for therapeutic interventions that leverage the positive modulation of the HGF pathway as a treatment for ALS.

Fosgonimeton, a Novel Positive Modulator of the HGF/MET System, Promotes Neurotrophic and Procognitive Effects in Models of Dementia.

All types of dementia, including Alzheimer's disease, are debilitating neurodegenerative conditions marked by compromised cognitive function for which there are few effective treatments. Positive modulation of hepatocyte growth factor (HGF)/MET, a critical neurotrophic signaling system, may promote neuronal health and function, thereby addressing neurodegeneration in dementia. Here, we evaluate a series of novel small molecules for their ability to (1) positively modulate HGF/MET activity, (2) induce neurotrophic changes and protect against neurotoxic insults in primary neuron culture, (3) promote anti-inflammatory effects in vitro and in vivo, and (4) reverse cognitive deficits in animal models of dementia. Through screening studies, the compound now known as fosgonimeton-active metabolite (fosgo-AM) was identified by use of immunocytochemistry to be the most potent positive modulator of HGF/MET and was selected for further testing. Primary hippocampal neurons treated with fosgo-AM showed enhanced synaptogenesis and neurite outgrowth, supporting the neurotrophic effects of positive modulators of HGF/MET. Additionally, fosgo-AM protected against neurotoxic insults in primary cortical neuron cultures. In vivo, treatment with fosgo-AM rescued cognitive deficits in the rat scopolamine amnesia model of dementia. Although fosgo-AM demonstrated several procognitive effects in vitro and in vivo, a prodrug strategy was used to enhance the pharmacological properties of fosgo-AM, resulting in the development of fosgonimeton (ATH-1017). The effect of fosgonimeton on cognition was confirmed in a lipopolysaccharide (LPS)-induced neuroinflammatory mouse model of dementia. Together, the results of these studies support the potential of positive modulators of HGF/MET to be used as novel therapeutics and suggest the drug candidate fosgonimeton might protect against neurodegeneration and be therapeutic in the management of Alzheimer's disease and other types of dementia.

Pericyte remodeling is deficient in the aged brain and contributes to impaired capillary flow and structure.

Deterioration of brain capillary flow and architecture is a hallmark of aging and dementia. It remains unclear how loss of brain pericytes in these conditions contributes to capillary dysfunction. Here, we conduct cause-and-effect studies by optically ablating pericytes in adult and aged mice in vivo. Focal pericyte loss induces capillary dilation without blood-brain barrier disruption. These abnormal dilations are exacerbated in the aged brain, and result in increased flow heterogeneity in capillary networks. A subset of affected capillaries experience reduced perfusion due to flow steal. Some capillaries stall in flow and regress, leading to loss of capillary connectivity. Remodeling of neighboring pericytes restores endothelial coverage and vascular tone within days. Pericyte remodeling is slower in the aged brain, resulting in regions of persistent capillary dilation. These findings link pericyte loss to disruption of capillary flow and structure. They also identify pericyte remodeling as a therapeutic target to preserve capillary flow dynamics.

Corrigendum: In vivo Single Cell Optical Ablation of Brain Pericytes.

[This corrects the article DOI: 10.3389/fnins.2022.900761.].

Imaging the construction of capillary networks in the neonatal mouse brain.

Capillary networks are essential for distribution of blood flow through the brain, and numerous other homeostatic functions, including neurovascular signal conduction and blood-brain barrier integrity. Accordingly, the impairment of capillary architecture and function lies at the root of many brain diseases. Visualizing how brain capillary networks develop in vivo can reveal innate programs for cerebrovascular growth and repair. Here, we use longitudinal two-photon imaging through noninvasive thinned skull windows to study a burst of angiogenic activity during cerebrovascular development in mouse neonates. We find that angiogenesis leading to the formation of capillary networks originated exclusively from cortical ascending venules. Two angiogenic sprouting activities were observed: 1) early, long-range sprouts that directly connected venules to upstream arteriolar input, establishing the backbone of the capillary bed, and 2) short-range sprouts that contributed to expansion of anastomotic connectivity within the capillary bed. All nascent sprouts were prefabricated with an intact endothelial lumen and pericyte coverage, ensuring their immediate perfusion and stability upon connection to their target vessels. The bulk of this capillary expansion spanned only 2 to 3 d and contributed to an increase of blood flow during a critical period in cortical development.

Three-dimensional ultrastructure of the brain pericyte-endothelial interface.

Pericytes and endothelial cells share membranous interdigitations called "peg-and-socket" interactions that facilitate their adhesion and biochemical crosstalk during vascular homeostasis. However, the morphology and distribution of these ultrastructures have remained elusive. Using a combination of 3D electron microscopy techniques, we examined peg-and-socket interactions in mouse brain capillaries. We found that pegs extending from pericytes to endothelial cells were morphologically diverse, exhibiting claw-like morphologies at the edge of the cell and bouton-shaped swellings away from the edge. Reciprocal endothelial pegs projecting into pericytes were less abundant and appeared as larger columnar protuberances. A large-scale 3D EM data set revealed enrichment of both pericyte and endothelial pegs around pericyte somata. The ratio of pericyte versus endothelial pegs was conserved among the pericytes examined, but total peg abundance was heterogeneous across cells. These data show considerable investment between pericytes and endothelial cells, and provide morphological evidence for pericyte somata as sites of enriched physical and biochemical interaction.

VasoMetrics: unbiased spatiotemporal analysis of microvascular diameter in multi-photon imaging applications.

BACKGROUND: Multi-photon imaging of the cerebrovasculature provides rich data on the dynamics of cortical arterioles, capillaries, and venules. Vascular diameter is the major determinant of blood flow resistance, and is the most commonly quantified metric in studies of the cerebrovasculature. However, there is a lack of accessible and easy-to-use methods to quantify vascular diameter in imaging data. METHODS: We created VasoMetrics, a macro written in ImageJ/Fiji for spatiotemporal analysis of microvascular diameter. The key feature of VasoMetrics is rapid analysis of many evenly spaced cross-sectional lines along the vessel of interest, permitting the extraction of numerous diameter measurements from individual vessels. Here we demonstrated the utility of VasoMetrics by analyzing in vivo multi-photon imaging stacks and movies collected from lightly sedated mice, as well as data from optical coherence tomography angiography (OCTA) of human retina. RESULTS: Compared to the standard approach, which is to measure cross-sectional diameters at arbitrary points along a vessel, VasoMetrics accurately reported spatiotemporal features of vessel diameter, reduced measurement bias and time spent analyzing data, and improved the reproducibility of diameter measurements between users. VasoMetrics revealed the dynamics in pial arteriole diameters during vasomotion at rest, as well as changes in capillary diameter before and after pericyte ablation. Retinal arteriole diameter was quantified from a human retinal angiogram, providing proof-of-principle that VasoMetrics can be applied to contrast-enhanced clinical imaging of microvasculature. CONCLUSIONS: VasoMetrics is a robust macro for spatiotemporal analysis of microvascular diameter in imaging applications.

Brain capillary pericytes exert a substantial but slow influence on blood flow.

The majority of the brain's vasculature is composed of intricate capillary networks lined by capillary pericytes. However, it remains unclear whether capillary pericytes influence blood flow. Using two-photon microscopy to observe and manipulate brain capillary pericytes in vivo, we find that their optogenetic stimulation decreases lumen diameter and blood flow, but with slower kinetics than similar stimulation of mural cells on upstream pial and precapillary arterioles. This slow vasoconstriction was inhibited by the clinically used vasodilator fasudil, a Rho-kinase inhibitor that blocks contractile machinery. Capillary pericytes were also slower to constrict back to baseline following hypercapnia-induced dilation, and slower to dilate towards baseline following optogenetically induced vasoconstriction. Optical ablation of single capillary pericytes led to sustained local dilation and a doubling of blood cell flux selectively in capillaries lacking pericyte contact. These data indicate that capillary pericytes contribute to basal blood flow resistance and slow modulation of blood flow throughout the brain.

Mild pericyte deficiency is associated with aberrant brain microvascular flow in aged PDGFRß+/- mice.

The receptor tyrosine kinase PDGFRß is essential for pericyte migration to the endothelium. In mice lacking one allele of PDGFRß (PDGFRß+/-), previous reports have described an age-dependent loss of pericytes in the brain, leading to cerebrovascular dysfunction and subsequent neurodegeneration reminiscent of that seen in Alzheimer's disease and vascular dementia. We examined 12-20-month-old PDGFRß+/- mice to better understand how pericyte loss affects brain microvascular structure and perfusion in vivo. We observed a mild reduction of cortical pericyte number in PDGFRß+/- mice (27% fewer cell bodies) compared to controls, but no decrease in pericyte coverage of the endothelium. This mild degree of pericyte loss caused no discernable change in cortical microvascular density, length, basal diameter or reactivity to hypercapnia. Yet, it was associated with an increase in basal blood cell velocity, primarily in pre-capillary arterioles. Taken together, our results suggest that mild pericyte loss can lead to aberrant cerebral blood flow despite a lack of apparent effect on microvascular structure and reactivity.

Organizational hierarchy and structural diversity of microvascular pericytes in adult mouse cortex.

Smooth muscle cells and pericytes, together called mural cells, coordinate many distinct vascular functions. Canonically, smooth muscle cells are ring-shaped and cover arterioles with circumferential processes, whereas pericytes extend thin processes that run longitudinally along capillaries. In between these canonical mural cell types are cells with features of both smooth muscle cells and pericytes. Recent studies suggest that these transitional cells are critical for controlling blood flow to the capillary bed during health and disease, but there remains confusion on how to identify them and where they are located in the brain microvasculature. To address this issue, we measured the morphology, vascular territory, and α-smooth muscle actin content of structurally diverse mural cells in adult mouse cortex. We first imaged intact 3D vascular networks to establish the locations of major gradations in mural cell appearance as arterioles branched into capillaries. We then imaged individual mural cells occupying the regions within these gradations. This revealed two transitional cells that were often similar in appearance, but with sharply contrasting levels of α-smooth muscle actin. Our findings highlight the diversity of mural cell morphologies in brain microvasculature, and provide guidance for identification and categorization of mural cell types.

Pericyte Structural Remodeling in Cerebrovascular Health and Homeostasis.

The biology of brain microvascular pericytes is an active area of research and discovery, as their interaction with the endothelium is critical for multiple aspects of cerebrovascular function. There is growing evidence that pericyte loss or dysfunction is involved in the pathogenesis of Alzheimer's disease, vascular dementia, ischemic stroke and brain injury. However, strategies to mitigate or compensate for this loss remain limited. In this review, we highlight a novel finding that pericytes in the adult brain are structurally dynamic in vivo, and actively compensate for loss of endothelial coverage by extending their far-reaching processes to maintain contact with regions of exposed endothelium. Structural remodeling of pericytes may present an opportunity to foster pericyte-endothelial communication in the adult brain and should be explored as a potential means to counteract pericyte loss in dementia and cerebrovascular disease. We discuss the pathophysiological consequences of pericyte loss on capillary function, and the biochemical pathways that may control pericyte remodeling. We also offer guidance for observing pericytes in vivo, such that pericyte structural remodeling can be more broadly studied in mouse models of cerebrovascular disease.

Dynamic Remodeling of Pericytes In Vivo Maintains Capillary Coverage in the Adult Mouse Brain.

Direct contact and communication between pericytes and endothelial cells is critical for maintenance of cerebrovascular stability and blood-brain barrier function. Capillary pericytes have thin processes that reach hundreds of micrometers along the capillary bed. The processes of adjacent pericytes come in close proximity but do not overlap, yielding a cellular chain with discrete territories occupied by individual pericytes. Little is known about whether this pericyte chain is structurally dynamic in the adult brain. Using in vivo two-photon imaging in adult mouse cortex, we show that while pericyte somata were immobile, the tips of their processes underwent extensions and/or retractions over days. The selective ablation of single pericytes provoked exuberant extension of processes from neighboring pericytes to contact uncovered regions of the endothelium. Uncovered capillary regions had normal barrier function but were dilated until pericyte contact was regained. Pericyte structural plasticity may be critical for cerebrovascular health and warrants detailed investigation.

Trial-unique, delayed nonmatching-to-location (TUNL) touchscreen testing for mice: sensitivity to dorsal hippocampal dysfunction.

RATIONALE: The hippocampus is implicated in many of the cognitive impairments observed in conditions such as Alzheimer's disease (AD) and schizophrenia (SCZ). Often, mice are the species of choice for models of these diseases and the study of the relationship between brain and behaviour more generally. Thus, automated and efficient hippocampal-sensitive cognitive tests for the mouse are important for developing therapeutic targets for these diseases, and understanding brain-behaviour relationships. One promising option is to adapt the touchscreen-based trial-unique nonmatching-to-location (TUNL) task that has been shown to be sensitive to hippocampal dysfunction in the rat. OBJECTIVES: This study aims to adapt the TUNL task for use in mice and to test for hippocampus-dependency of the task. METHODS: TUNL training protocols were altered such that C57BL/6 mice were able to acquire the task. Following acquisition, dysfunction of the dorsal hippocampus (dHp) was induced using a fibre-sparing excitotoxin, and the effects of manipulation of several task parameters were examined. RESULTS: Mice could acquire the TUNL task using training optimised for the mouse (experiments 1). TUNL was found to be sensitive to dHp dysfunction in the mouse (experiments 2, 3 and 4). In addition, we observed that performance of dHp dysfunction group was somewhat consistently lower when sample locations were presented in the centre of the screen. CONCLUSIONS: This study opens up the possibility of testing both mouse and rat models on this flexible and hippocampus-sensitive touchscreen task.