Deregulation of the Nerve Growth Factor Pathway in Primary Cultures from Down Syndrome Fetal Cortex
Alison Kate Ower
Supervisor: Dr. Claudio Cuello
Due to triplication of chromosome 21, by middle age most individuals with Down’s syndrome (DS) will develop the neuropathological changes characteristic of Alzheimer’s disease (AD). In AD there is an early atrophy of basal forebrain cholinergic neurons (BFCN), a neuronal network with key roles in learning and memory. These cells depend on the endogenous supply of nerve growth factor (NGF) throughout life. The Cuello lab has previously demonstrated that the maturation and degradation of this neurotrophin are deregulated in AD brains.
Due to the presence of AD pathology in DS brains, we hypothesized that a similar imbalance should be present in this condition. Therefore, this project sought to investigate the presence of alterations in the metabolism of NGF, in an early, fetal stage of DS. We used primary cultures generated from DS fetal cortex (17-21 gestational age weeks) to examine the levels and activity of different players within the NGF metabolism pathway.
As expected due to a triplication of the gene for amyloid precursor protein (APP) on chromosome 21, we observed an increase in soluble APP in both cellular lysates and in conditioned media. We also observed increased levels of secreted beta-amyloid peptides in conditioned media from DS cultures at this early stage. We observed a significant increase in the pro-form of NGF in DS conditioned media relative to controls, and decreased activity of tissue plasminogen activator (tPA), a protein responsible for regulating the enzyme controlling the conversion of the pro-form to its mature state (see Figure 1). Previous results from the Cuello lab have demonstrated matrix metalloprotease-9 (MMP-9) activity to be increased in AD brains. MMP-9 is responsible for the degradation of the mature form of NGF in the extracellular space. We were able to detect increased MMP-9 activity in DS conditioned media, as well as a corresponding increase in the protein levels MMP-9. The cultures with the highest MMP-9 activity also had higher levels of MMP-9 protein. The accumulation of proNGF and the increased activity of MMP-9 in DS cultures suggest that the maturation and degradation of NGF are altered in DS fetal tissue. These changes have a potential negative effect on the phenotypic maintenance of BFCN. The alterations of key proteins related to NGF metabolism are shown in Table 1.
One of the most significant findings of this research is the ability to detect alterations to this pathway in a human foetal stage. These findings support the concept of a role of amyloid accumulation in the dysmetabolism of NGF. The ability to detect alterations in NGF metabolism at such an early stage of disease in humans suggests their potential use as biomarkers of disease. The development of a biomarker based on these findings would help determine whether or not alterations linked to developing AD are present in patients who have not already shown clinical evidence of dementia.
Our findings suggest that early amyloid pathogenesis could deregulate NGF metabolism before the presence of full-blown Alzheimer’s disease pathological hallmarks. Current treatments for AD have not been shown to alter the final outcome of the disease. One hope is that new drugs could target mechanisms that would modify the progression of the disease. The alterations of the NGF metabolic pathway could provide a new paradigm for drug development to treat AD in DS.
Figure 1 ProNGF is converted to mature NGF (mNGF) by plasmin, which derives from plasminogen by the action of tissue plasminogen activator (tPA). MMP-9 is the main protease responsible for mNGF degradation. Modified from Bruno and Cuello, PNAS 2006.