Z-Phe-Ala-diazomethylketone

Accumulating evidence suggests that the endolysosomal system is a novel intracellular Ca(2+) pool mobilized by the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). Although lysosomes in neurons are known to proliferate in numerous neurodegenerative diseases and during the normal course of aging, little is known concerning the effect of lysosomal proliferation on Ca(2+) homeostasis. Here, we induce proliferation of lysosomes in primary cultures of rat hippocampal neurons and PC12 cells through chronic treatment with the cathepsin inhibitor, Z-Phe-Ala-diazomethylketone. We demonstrate that lysosome proliferation increases the size of the lysosomal Ca(2+) pool and enhances Ca(2+) signals in response to direct cellular delivery of NAADP and glutamate, an identified NAADP-producing agonist. Our data suggest that deregulated lysosomal Ca(2+) signaling through NAADP may contribute to neuronal dysfunction and highlight the usefulness of lysosomal hydrolase inhibition in probing NAADP action.

Many neurodegenerative disorders have lysosomal impediments, and the list of proposed treatments targeting lysosomes is growing. We investigated the role of lysosomes in Alzheimer's disease (AD) and other age-related disorders, as well as in a strategy to compensate for lysosomal disturbances. Comprehensive immunostaining was used to analyze brains from wild-type mice vs. amyloid precursor protein/presenilin-1 (APP/PS1) mice that express mutant proteins linked to familial AD. Also, lysosomal modulation was evaluated for inducing synaptic and behavioral improvements in transgenic models of AD and Parkinson's disease, and in models of mild cognitive impairment (MCI). Amyloid plaques were surrounded by swollen organelles positive for the lysosome-associated membrane protein 1 (LAMP1) in the APP/PS1 cortex and hippocampus, regions with robust synaptic deterioration. Within neurons, lysosomes contain the amyloid β 42 (Aβ42) degradation product Aβ38, and this indicator of Aβ42 detoxification was augmented by Z-Phe-Ala-diazomethylketone (PADK; also known as ZFAD) as it enhanced the lysosomal hydrolase cathepsin B (CatB). PADK promoted Aβ42 colocalization with CatB in lysosomes that formed clusters in neurons, while reducing Aβ deposits as well. PADK also reduced amyloidogenic peptides and α-synuclein in correspondence with restored synaptic markers, and both synaptic and cognitive measures were improved in the APP/PS1 and MCI models. These findings indicate that lysosomal perturbation contributes to synaptic and cognitive decay, whereas safely enhancing protein clearance through modulated CatB ameliorates the compromised synapses and cognition, thus supporting early CatB upregulation as a disease-modifying therapy that may also slow the MCI to dementia continuum.

The oligomerization of the amyloid-β protein (Aβ) is an important event in Alzheimer disease (AD) pathology. Developing small molecules that disrupt formation of early oligomeric states of Aβ and thereby reduce the effective amount of toxic oligomers is a promising therapeutic strategy for AD. Here, mass spectrometry and ion mobility spectrometry were used to investigate the effects of a small molecule, Z-Phe-Ala-diazomethylketone (PADK), on the Aβ42 form of the protein. The mass spectrum of a mixture of PADK and Aβ42 clearly shows that PADK binds directly to Aβ42 monomers and small oligomers. Ion mobility results indicate that PADK not only inhibits the formation of Aβ42 dodecamers, but also removes preformed Aβ42 dodecamers from the solution. Electron microscopy images show that PADK inhibits Aβ42 fibril formation in the solution. These results are consistent with a previous study that found that PADK has protective effects in an AD transgenic mouse model. The study of PADK and Aβ42 provides an example of small molecule therapeutic development for AD and other amyloid diseases.