[Mechanisms of deregulated response to hypoxia in sporadic amyotrophic lateral sclerosis: a clinical study]. / Mécanismes de dérégulation de la réponse à l'hypoxie dans la sclérose latérale amyotrophique.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder of upper and lower motorneurons, leading to death in 3 to 5 years. Respiratory insufficiency and hypoxemia are closely linked during the clinical course of ALS. Chronic respiratory insufficiency and hypoxemia generally occur late in the disease course but rapid episodes of intermittent hypoxemia followed by reoxygenation can occur early and insidiously. Two pathways are involved in the response to hypoxemia: (i) hypoxia inducible factor-1 (HIF-1) and VEGF/HIF-2 and an erythropoietin (EPO) mediated pathway, in response to prolonged hypoxemia; and (ii) nuclear factor kappa-B (NFkappa-B) during acute hypoxemia followed by reoxygenation episodes, inducing inflammatory mediators: interleukin-6 (IL-6), TNF-alpha, cyclo oxygenase-2 (COX-2) and prostaglandin E-2 (PGE-2). Our aim was to specify the role of the different functional pathways of response to hypoxemia in sporadic ALS patients, compared with neurological controls and according to the level of hypoxemia. We report the results of several studies of hypoxemic and/or inflammatory mediators in the cerebrospinal fluid (CSF) from ALS patients, according to their respiratory status, showing a selective defect of HIF-1 mediated angiogenic factors (VEGF and angiogenin [ANG]) during chronic hypoxia in sporadic ALS patients, compared to hypoxemic neurological controls; contrasting with an early activation of the NFkappa-B pathway since the isolated desaturation stage (IL-6, TNF-alpha, PGE-2, angiopoietin-2) in the same cohort of sporadic ALS patients. All these results are consistent with a selective impairment of the HIF-1 pathway during chronic hypoxemia in ALS patients. Inflammatory mediators were strongly elevated, since the early stage of the disease until chronic hypoxemia, suggesting a compensatory mechanism.

High erythropoietin and low vascular endothelial growth factor levels in cerebrospinal fluid from hypoxemic ALS patients suggest an abnormal response to hypoxia.

Animal studies have highlighted the potentially neuroprotective role of vascular endothelial growth factor (VEGF). Low levels of this growth factor have been found in the cerebrospinal fluid (CSF) of patients with amyotrophic lateral sclerosis (ALS). VEGF (and other proteins, such as erythropoietin (EPO)) are produced in response to hypoxia via a common pathway involving a specific transcription factor (hypoxia-inducible factor, HIF) and a hypoxia responsive element (HRE) in the respective genes' promoter regions. In this study, we report finding the expected, high levels of VEGF and EPO in CSF from hypoxemic neurological controls, whereas EPO (but not VEGF) levels are high in the CSF from hypoxemic ALS patients. Hence, the VEGF levels in CSF from patients with ALS were significantly lower than those seen in hypoxemic controls. There was a trend towards higher CSF levels of EPO in hypoxemic ALS patients than in hypoxemic controls. Our results suggest that VEGF may not be produced in sufficient amounts in chronically hypoxic ALS patients and that this dysfunction may participate in the pathogenesis of the disease. The high EPO levels in hypoxemic ALS patients nevertheless suggest an intact common oxygen-sensor pathway.

Deregulation of the hypoxia inducible factor-1α pathway in monocytes from sporadic amyotrophic lateral sclerosis patients.

The clinical course of the degenerative motor neuron disorder amyotrophic lateral sclerosis (ALS) is closely related to hypoxia. The normal response to hypoxia involves two pathways in particular: the hypoxia inducible factor 1α (HIF-1α) pathway (which notably controls the synthesis of vascular endothelial growth factor (VEGF)) and the nuclear factor kappa B (NF-κb) pathway (responsible for the production of inflammatory mediators, including prostaglandin E2 (PGE2)). Defects in VEGF gene expression are known to cause motor neuron degeneration in animal models. Circulating monocytes are precursors of the microglia, which are involved in the pathogenesis of ALS. To establish whether the HIF-1 and/or NF-κB pathways are deregulated during hypoxia in early-stage, sporadic ALS, we analyzed the response to acute (1 h) and prolonged (24 h) hypoxia in monocytes from ALS and healthy controls. We measured protein expression and mRNA transcription for VEGF, HIF-1, HIF-2, prolyl hydroxylases 1 and 2 (PHD-1 and -2, part of the HIF proteasome-dependent degradation pathway) and their modulation by PGE2. Our results showed that (i) the HIF-1 (but not HIF-2) and VEGF production induced by acute and prolonged hypoxia was selectively and markedly altered in ALS patients and (ii) this defect was not compensated for by PGE2 addition. Moreover, altered HIF-1α activation was associated with low levels of proteolysis by PHD-2 in cells from sporadic ALS patients (relative to controls). For the first time, we have demonstrated clinical and functional abnormalities in the HIF-1 pathway during hypoxia in monocytes from sporadic ALS patients.

Paradoxical response of VEGF expression to hypoxia in CSF of patients with ALS.

Vascular endothelial growth factor (VEGF) is implicated in motor neurone degeneration. In normal individuals, hypoxia is known to induce an overexpression of VEGF, as measured in CSF. We show that patients with ALS do not manifest this VEGF overexpression in the presence of hypoxia. Although VEGF gene expression is mainly stimulated by hypoxia, we have measured lower VEGF levels in cerebrospinal fluid (CSF) from hypoxaemic patients with amyotrophic lateral sclerosis (ALS) than in CSF from normoxaemic patients with ALS. In contrast, hypoxaemic neurological controls displayed higher levels than normoxaemic neurological controls. There was a negative correlation between VEGF levels and the severity of hypoxaemia in patients with ALS, suggesting deregulation of VEGF in ALS.

Elevated IL-6 and TNF-alpha levels in patients with ALS: inflammation or hypoxia?

Abnormal levels of interleukin (IL)-6 were described in patients with ALS, related to an inflammatory process. The authors compared IL-6 and tumor necrosis factor alpha (TNF-alpha) levels in CSF and sera from 10 hypoxemics and 10 normoxemics patients with ALS to those of 10 hypoxemics and 10 normoxemics neurologic controls. The same pattern exists in patients with ALS and controls: the highest levels are found in hypoxic conditions and undetectable levels are found in normoxemic conditions. Elevated IL-6 levels in ALS could correspond to a normal response to hypoxemia.

Low levels of the vascular endothelial growth factor in CSF from early ALS patients.

Deletion of the hypoxia-response element in the vascular endothelial growth factor (VEGF) promoter causes motor neuron degeneration in a mouse model. "At-risk" haplotypes with low circulating VEGF levels have been demonstrated in humans. Here the authors report low VEGF levels in the CSF of ALS patients during their first year of the disease, independently of VEGF promoter polymorphism. This finding early in ALS patients suggests a possible role for VEGF gene regulation in the pathogenesis of ALS.