| Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive and fatal motor neuron disease, characterized by loss of motor neurons in the cortex, brainstem and spinal cord. Clinical management is plagued by a lack of biomarkers and effective treatment. In spite of numerous scientific advancements, molecular mechanisms involved in its initiation and progression remain an enigma. At the mechanistic level, ALS is considered multifactorial. Extracellular signals may modulate nuclear events with a possible consequence being the reactivation of cell cycle-related genes and protein alterations in the terminally differentiated motor neurons.;In the first specific aim, we hypothesized that re-entry of post-mitotic motor neurons into the cell cycle, concurrent with altered activity or distribution of transcription factors will result in apoptosis of motor neurons during ALS. To address this hypothesis, we utilized archived human autopsy material from the cortical and spinal cord regions of ALS and age-matched control cases. We conclude that surviving ALS motor neurons in these regions exhibited increased levels of G1 to S phase regulators (Cyclin D1, CDK4, hyperphosphorylated-pRb and E2F-1). It also revealed two intriguing results; (i) E2F-1, a transcription factor, was cytoplasmic and (ii) increased nuclear p53 was noted in spinal motor neurons but absent in neurons of the motor cortex. In addition there was increased protein levels of apoptotic death markers (BAX, FAS, Caspases) and DNA fragmentation. Therefore we have identified a potential role for cell cycle proteins in an apoptotic mode of motor neuron death in ALS.;In the second specific aim we hypothesized that a mass spectrometry-based proteomics approach will identify diagnostic biomarkers and molecular targets for drug discoveries. We used cerebrospinal fluid (CSF) from ALS and control subjects to identify and validate a biomarker panel specific to ALS. Furthermore, utilizing peptide map fingerprinting and tandem mass-spectrometry, we have identified three of the protein peaks to be a carboxyl-terminal fragment of neurosecretory chaperone protein 7B2 (3.44kDa), Cystatin C (13.3kDa) and monomer of transthyretin (13.78kDa).;Taken together, this body of work furthers the understanding of both the mechanisms leading to selective motor neuron loss in ALS and paves the way for diagnostics and therapeutics. |
