Hemozoin induces neuronal injury primarily characterized by axon rupture and mitochondrial damage in experimental cerebral malaria

TL;DR

This study shows that hemozoin, a byproduct of malaria parasites, directly harms neurons by damaging axons and mitochondria, leading to brain injury in cerebral malaria.

Contribution

The study is the first to demonstrate that hemozoin directly adheres to and is internalized by neurons, causing structural and mitochondrial damage.

Findings

Hemozoin adheres to neurons and is internalized into their cytoplasm, causing axon rupture and plasma membrane disruption.

Hemozoin induces mitochondrial dysfunction, reduced ATP levels, and increased reactive oxygen species in neurons.

Hemozoin upregulates neuronal nitric oxide synthase activity and decreases neurotransmitter levels like glutamate.

Abstract

Cerebral malaria (CM) is the most serious and fatal neurological complication of Plasmodium falciparum infection, which can cause death or long-term neurological sequelae. Neuronal injury is a primary cause of these sequelae in patients with CM; however, the underlying mechanisms remain incompletely elucidated. Hemozoin (Hz), the metabolic byproduct of hemoglobin digested by Plasmodium parasites, is closely associated with the severity of CM. However, it is not clear whether Hz is a direct contributor to neuronal injury.

C57BL/6 J mice were infected with the Plasmodium berghei ANKA (PbA) strain to induce experimental cerebral malaria (ECM). Hz deposition and neuronal injury in ECM mice brain tissues were assessed using histopathological staining. In vitro, primary cortical neurons were stimulated with purified hemozoin (pHz). Neuronal morphology, pHz internalization, and injury severity were assessed via transmission electron microscopy (TEM), live-cell imaging, and lactate dehydrogenase (LDH) assays, respectively. Furthermore, Mito-Tracker and JC-1 probes were used to analyze mitochondrial content and membrane potential, respectively. ATP assay kits were used to quantify cellular energy metabolism levels, while reactive oxygen species (ROS)/neuronal nitric oxide synthase (nNOS) fluorescent probes were used to assess oxidative stress and inflammatory response. Neurotransmitter alterations were analyzed by measuring glutamate (Glu) levels.

In the cerebral cortex of ECM mice, significant Hz deposition and reduced neuronal nuclei (NeuN) expression levels were observed. Immunofluorescence (IF) staining demonstrated that pHz adhered to primary neurons in vitro, causing reduced dendritic arborization, axon rupture, and plasma membrane disruption. TEM and live-cell imaging confirmed that pHz was internalized into the cytoplasm of neurons. Furthermore, pHz induced mitochondrial structural damage and reduced mitochondrial content. Concurrently, pHz triggered mitochondrial dysfunction, characterized by diminished mitochondrial membrane potential (MMP), reduced ATP levels, and elevated ROS. In addition, pHz upregulated intraneuronal nNOS activity and caused a decrease in neurotransmitter levels.

This study provided the first evidence to our knowledge that Hz directly adhered to neurons and underwent internalization into its cytoplasm, thereby leading to neuronal injury. These findings elucidate a potential mechanism underlying neuronal injury in ECM and inform the development of adjuvant therapies targeting Hz.

The online version contains supplementary material available at 10.1186/s13071-025-07102-5.

Figures