The top portion of this graphic features Neurons (blue) with axons wrapped by normal myelin (yellow). The lower portion of this graphic illustrates neurons with pathological axons wrapped by damaged myelin (yellow and pink). The elongated mitochondria (purple) in the lower portion are dysfunctional and characterized by accumulation of toxic ceramides (green) [Jeremy Weichsel at Biovisioning]

Multiple sclerosis (MS) is a neurodegenerative disorder that may take two basic forms, relapsing remitting MS (RMMS), which presents with periods of clinical remission, and progressive MS, which is characterized by continued deterioration without remission. There are some therapies available to help manage RRMS, but treating progressive MS is far more challenging. By studying the effects of cerebrospinal fluid (CSF) from MS patients on mitochondria in mouse neurons, U.S. researchers have now identified a biological mechanism that might ultimately help develop new therapeutic strategies against the progressive form of the disease.

“Because the brain is bathed by the CSF, we asked whether treating cultured neurons with the CSF from MS patients with a relapsing/remitting or a progressive disease course would possibly elicit different effects on neuronal mitochondrial function,” said the study’s primary investigator Patrizia Casaccia, PhD, Einstein professor of biology at the Graduate Center and founding director of the Neuroscience Initiative at the Advanced Science Research Center (ASRC) at the Graduate Center, City University of New York, and the Icahn School of Medicine at Mount Sinai. “We detected dramatic differences in the shape of the neuronal mitochondria and their ability to produce energy.”

Casaccia and colleagues reported their findings in Brain, in a paper titled, “A metabolic perspective on CSF-medicated neurodegeneration in multiple sclerosis.”

MS affects more than 2.5 million people worldwide, and is characterized by destruction of the myelin sheath that surrounds nerve cells. RRMS is the most common form of MS and affects about 85% of patients, who exhibit demyelinating inflammatory episodes with clinical symptoms, followed by periods of clinical remission. The 15% of patients who present with primary progressive MS exhibit progressive neurological deterioration without periods of clinical remission. Approximately 50% of RRMS patients will also eventually develop progressive disease.

While there are approved immunomodulatory drugs that can help decrease inflammation that is characteristic of RRMS, “progressive MS, with disability driven by neurodegeneration and inflammation that is intrinsic to the CNS, has been more difficult to manage,” the authors noted. “One of the greatest challenges for the field of MS remains the therapeutic management of the neurodegenerative component of the disease. This is likely due to the elusive nature of the molecular mechanisms underlying disease progression, which has precluded the potential definition of effective therapeutic target.”

Previous studies in animals have suggested that dysfunction of mitochondria in nerve cells may be a feature of progressive MS, but the molecular mechanisms underlying the process aren’t known. To look at this in more detail Casaccia and colleagues investigated whether there were any differential effects of treating rat neurons with CSF taken from human RRMS patients or those with progressive MS.

The researchers functionally and metabolically characterized CSF samples from 15 patients with RRMS and another 29 with progressive MS, and exposed live cultured rat neurons to the samples. Any effects on the neurons were recorded directly by time-lapse videos using live confocal imaging. A mitochondrial tracer was used to allow visualization of any changes to mitochondria. The videos revealed important differences between the effects of the two different CSF sample types. Mitochondria exposed to CSF from progressive MS patients became much more elongated and fused together. “Notably, we detected a substantial elongation of these organelles, coalescing to form a tubular network only in neuronal cultures exposed to the CSF from progressive patients,” the team reported. This response was not seen in mitochondria exposed to CSF from patients with a relapsing/remitting MS.

Further biochemical tests showed that the elongated mitochondria didn’t function as well and so were less capable of producing energy, which eventually resulted in neuronal cell death. “We detected dramatic differences in the shape of the neuronal mitochondria and their ability to produce energy,” Casaccia stated. “Only exposure to the CSF from progressive MS patients caused neuronal mitochondria to fuse and elongate while rendering them unable to produce energy.”

Previous research has suggested that mitochondria elongate in an attempt to generate more energy for cells when there is enhanced energetic demand or a decrease in available glucose. To try and find what might be present in the CSF of progressive MS that triggers this elongation response, the team first destroyed any proteins in the samples by subjecting them to heat, and then retested the heat-treated samples on rat neurons. Interestingly, there was still a “remarkable effect” of the CSF from progressive patients on mitochondrial elongation, which the researchers say “ruled out a potential contribution of protein components.”

They then carried out an analysis of lipid components in the CSF from RRMS patients and from progressive MS patients, and found increased levels of ceramides, and particularly ceramide C24, in the progressive MS CSF. Ceramides are sphingolipids that have previously been implicated in MS, the authors pointed out.

Significantly, exposing rat neurons to ceramides resulted in the same mitochondrial elongation as had exposure to the progressive MS CSF. “When we exposed cultured neurons to ceramides, we elicited the same changes caused by exposure to CSF from progressive MS patients,” said Maureen Wentling, PhD, a research associate in the Casaccia lab and the study’s first author.

Further studies in cultured neurons exposed to ceramide either in conditions of low or high glucose indicated that the treatment impairs ATP production. “… the presence of ceramides interferes with the activity of respiratory chain complexes which become dysfunctional,” the investigators stated. “The neuron attempts to compensate this energetic deficit by upregulating glucose transporters and re-directing the energetic response towards glycolysis in an attempt to meet the metabolic demand, which in the long terms proves to be inefficient and lead to neurotoxicity.”

“… we further discovered that ceramides induced neuronal damage by acting on two cellular mechanisms,” Wentling added. “On one end, ceramides impaired the ability of neurons to make energy by directly damaging the mitochondria. On the other end, they also forced neurons to more rapidly uptake glucose in an attempt to provide energy for the cell.”

The neurotoxic effects of CSF on cultured neurons could be reduced by supplementing the neurons with glucose or lactate. Although this approach wouldn’t work as a sustainable therapeutic strategy, the results may help researchers develop new approaches to protect mitochondria in patients with progressive MS, while ceramides in CSF may represent potential biomarkers of neurodegeneration. “Together these data suggest a condition of ‘virtual hypoglycosis’ induced by the CSF of progressive patients in cultured neurons and suggest a critical temporal window of intervention for the rescue of the metabolic impairment of neuronal bioenergetics underlying neurodegeneration in MS patients,” the researchers concluded. “The role of specific CSF ceramides as potential biomarkers for neurodegeneration is also of great interest and awaits further validation in larger cohorts of MS patients, in future studies.”

 

 

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