Peter Lobel
	Professor Department of Pharmacology UMDNJ-Robert Wood Johnson Medical School Member Cancer Institute of New Jersey Ph.D., 1986, Columbia University Tel:  [732] 235-5032 Fax: [732] 235-5289 lobel@cabm.rutgers.edu

Lysosomal storage disorders, neuronal ceroid lipofuscinosis, Niemann Pick disease, proteomics, mannose 6-phosphate receptors.

Our laboratory has developed new methods for disease discovery and identified the molecular bases for three fatal neurodegenerative disorders. This work grew out of our basic research on lysosomal enzyme targeting.

Lysosomes are membrane-bound, acidic organelles that are found in all eukaryotic cells. They contain a variety of different proteases, glycosidases, lipases, phosphatases, nucleases and other hydrolytic enzymes, most of which are delivered to the lysosome by the mannose 6-phosphate targeting system. In this pathway, lysosomal enzymes are recognized as different from other glycoproteins and are selectively phosphorylated on mannose residues. The mannose 6-phosphate serves as a recognition marker that allows the enzymes to bind mannose 6-phosphate receptor which ferry the lysosomal enzyme to the lysosome. In the lysosome, the enzymes function in concert to break down complex biological macromolecules into simple components. The importance of these enzymes is underscored by the identification of over thirty lysosomal storage disorders (e.g., Tay Sach's disease) where loss of a single lysosomal enzyme leads to severe health problems including neurodegeneration, progressive mental retardation, and early death. There are also a number of unsolved genetic diseases that are likely to arise from deficiencies in as yet undiscovered lysosomal enzymes.

Our approach to identify the molecular basis for unsolved lysosomal storage disorders is based on our ability to use mannose 6-phosphate receptor derivatives to visualize and purify mannose 6-phosphate containing lysosomal enzymes. For instance, we can fractionate proteins in normal and disease specimens by 2-dimensional gel eletecrophoresis and then, in a manner analogous to Western blotting, use a radiolabeled mannose 6-phosphate receptor derivative to selectively visualize phosphorylated lysosomal enzymes. This allows us to compare the spectrum of lysosomal enzymes present in normal and disease specimens. If the disease specimen lacks a given lysosomal protein, this may be responsible for disease. To investigate this, we purify and sequence the normal protein, clone the corresponding gene, and examine patients for mutations associated with disease. In this manner, we found that a fatal childhood neurodegenerative disease called LINCL (late infantile neuronal ceroid lipofuscinosis) is caused by mutations in a gene encoding a previously undiscovered lysosomal protease.

LINCL is literally a disease from hell, as parents see what has been a normally developing child degenerate before their eyes. Children typically develop normally until age 3 at which point they exhibit ataxia and seizures. They start losing vision a year later, and within a few years are blind, mute, and completely bedridden. The children usually die between ages eight and fifteen, although there are some mutations that result in a later-onset, prolonged disease. At the cellular level there is extensive lysosomal accumulation of autofluorescent storage material (ceroid lipofuscin) accompanied by massive death of neurons and marked brain atrophy. About fifty children are diagnosed with LINCL each year in the United States and the disease has devastating effects on the affected children and families.

While our laboratory primarily conducts basic research, our interactions with many LINCL families have given us added impetus to extend our research to the clinic. After we identified the gene and determined the function of corresponding protein, we developed rapid biochemical and DNA-based assays for definitive pre-and postnatal diagnosis and carrier screening. This allows for genetic counseling to prevent further occurrence of the disease. However, in the absence of universal carrier testing, new cases will continue to arise so it is important to develop effective therapies that can halt and reverse disease progression. To this end, we have produced recombinant enzyme in a form that can be taken up by affected cells in culture to correct the primary defect. We are also working to develop a LINCL mouse model that should allow detailed studies of disease pathophysiology and evaluation of potential therapeutics strategies.

Another research program in the laboratory is to identify the spectrum of lysosomal enzymes encoded by the human genome. This research is particularly timely given the current effort towards determining the complete sequence of the human genome. Our approach is to purify mannose 6-phosphorylated proteins by affinity chromatography, resolve the mixture by two dimensional gel electrophoresis, and then analyze each protein by peptide mapping, mass spectrometry, and chemical sequencing. This information is used to search sequence databases to determine if a given protein corresponds to a known lysosomal enzyme or if it represents a previously unidentified species. We have currently identified a number of new lysosomal proteins and are working to characterize their role in biology and medicine. We recently used this approach to determine the molecular basis for Niemann Pick type C2 disease, a fatal cholesterol storage disorder.

In addition to their roles in human inherited diseases, alterations in the lysosomal system have been implicated in a variety of disease processes such as tumor invasion and metastasis in cancer, tissue destruction in arthritis, and early changes associated with Alzheimer disease. Once we develop the tools to visualize and characterize the players, our ultimate goal will be to understand the role that lysosomal proteins play in these widespread pathological processes.

Identification of a lysosomal protein deficient in a hereditary human neurodegenerative disease. Specimens from an unaffected control and a child diagnosed with late infantile neuronal ceroid lipofuscinosis were fractionated by two dimensional gel electrophoresis, transferred to a membrane, and probed with radiolabeled mannose 6-phosphate receptor. This revealed the absence of a specific protein in the disease specimen and lead to the identification of the molecular basis of the disease.

Selected Publications1