Neuronal Ceroid Lipofuscinosis are a group of devastating neurodegenerative lysosomal storage disorders that begin in childhood. Mutations in CLN3 gene lead to a NCL called Batten disease, characterized by the progressive loss of vision, movement, and cognition. Targeted effective therapies are not available for these disorders since the biological roles of most genes responsible for these disorders are not well defined.

Researchers at Baylor College of Medicine and Texas Children's Hospital in the US, Telethon Institute of Genetics and Medicine, and Federico II University in Italy have now discovered that CLN3 is critical for lysosome biogenesis and autophagic lysosomal reformation (ALR), uncovering a novel disease mechanism in Batten disease.

Lysosomes act as cellular digestors and compactors by breaking down a wide range of cellular polymers and debris. Lysosomal biogenesis is a key process required for the de-novo ('from scratch') generation of lysosomes, while ALR is a terminal step of autophagy, a cellular degradative process induced under starvation or other stress conditions.

Lysosomes are consumed during the initial steps of autophagy and during times of prolonged starvation are generated back via ALR, a process by which new functional lysosomes are formed from mature lysosomes. Here, the team found CLN3 is critical for the generation of new lysosomes, by impairing both de novo lysosomal biogenesis and ALR pathways, which results in the accumulation of "aged" lysosomes that are unable to function properly.

The study, published in Nature Communications, was led by Dr. Andrea Ballabio, professor at Baylor College and principal investigator at the Jan and Dan Duncan Neurological Research Institute (Duncan NRI), and Dr. Alessia Calcagni, assistant professor at Baylor College.

"Given how important renewal of lysosomes and autophagy is for cellular survival and function, this study has potentially far-reaching implications not just for Batten disease but for many other conditions that are caused by defects in lysosomal trafficking and storage," Dr. Ballabio said.

CLN3 transits through the Golgi complex to reach the lysosomes

The lack of a reliable tool to study CLN3 had been an obstacle to the researchers studying Batten disease and so, the team first generated a novel CLN3 antibody.

To their surprise, they found CLN3 was not just present in the lysosomes as was thought previously but that when first synthesized, it transits through the Golgi complex where it acquires several modifications before it reaches its final destination, the lysosomes.

Loss of CLN3 results in enlarged non-functional lysosomes

Next, to explore the biological role(s) of CLN3, they performed an interactome analysis to identify all the proteins that bind to CLN3. These experiments revealed that several of its partners are proteins involved in the intracellular trafficking pathways and formation of new lysosomes, suggesting that it may play an important role in these processes.

A lysosomal sorting protein, cation-independent mannose-6-phosphate receptor (CI-M6PR) was found to be among the most enriched interactors of CLN3. Most lysosomal enzymes acquire mannose-6-phosphate residues as an "address label" which are then recognized by specific mannose-6-phosphate receptors (analogous to a "delivery person") to be routed to the correct endo-lysosomal compartments ("destination").

Interestingly, they found that the loss of CLN3 results in mis-sorting of CI-M6PR itself to the lysosomes where it was degraded. As expected, this disrupted lysosomal biogenesis. The new lysosomes showed a global depletion of several degradative enzymes. In addition, the absence of CLN3 resulted in enlarged lysosomes and the aggregation of various intermediates involved in autophagy, which is a feature of impaired ALR and lysosomal function.

The lysosomal degradative defects caused by the mis-sorting of CI-M6PR in CLN3-depleted cells, abolished both initial consumption of lysosomes and their reformation during starvation, blocking ALR and thus, autophagy.

CI-M6PR is essential for CLN3-mediated ALR

When the researchers overexpressed CLN3 in cells lacking the CLN3 gene, it resulted in a substantial increase in the number of lysosomes, particularly during prolonged starvation, reduced size of the lysosomes, increased the levels of lysosomal enzymes, and promoted the autophagy-dependent formation of new lysosomes. Conversely, silencing CI-M6PR completely abolished CLN3-mediated tubulation and reformation of lysosomes.

"We hope knowing exactly how CLN3 affects lysosomal function will help us and others identify effective therapies for Batten disease in the future," Dr. Alessia Calcagni said. "Moreover, this study indicates ALR impairment and a global reduction in the degradative capacity of lysosomes could be a possible underlying cause for other NCLs and will be important to test in the future."