Concomitant gain and loss of function pathomechanisms in C9ORF72 amyotrophic lateral sclerosis.

Intronic hexanucleotide repeat expansions (HREs) in C9ORF72 are the most frequent genetic cause of amyotrophic lateral sclerosis, a devastating, incurable motoneuron (MN) disease. The mechanism by which HREs trigger pathogenesis remains elusive. The discovery of repeat-associated non-ATG (RAN) translation of dipeptide repeat proteins (DPRs) from HREs along with reduced exonic C9ORF72 expression suggests gain of toxic functions (GOFs) through DPRs versus loss of C9ORF72 functions (LOFs). Through multiparametric high-content (HC) live profiling in spinal MNs from induced pluripotent stem cells and comparison to mutant FUS and TDP43, we show that HRE C9ORF72 caused a distinct, later spatiotemporal appearance of mainly proximal axonal organelle motility deficits concomitant to augmented DNA double-strand breaks (DSBs), RNA foci, DPRs, and apoptosis. We show that both GOFs and LOFs were necessary to yield the overall C9ORF72 pathology. Increased RNA foci and DPRs concurred with onset of axon trafficking defects, DSBs, and cell death, although DSB induction itself did not phenocopy C9ORF72 mutants. Interestingly, the majority of LOF-specific DEGs were shared with HRE-mediated GOF DEGs. Finally, C9ORF72 LOF was sufficient-albeit to a smaller extent-to induce premature distal axonal trafficking deficits and increased DSBs.

Generation and deposition of Aß43 by the virtually inactive presenilin-1 L435F mutant contradicts the presenilin loss-of-function hypothesis of Alzheimer's disease.

As stated by the prevailing amyloid cascade hypothesis, Alzheimer's disease (AD) is caused by the aggregation and cerebral deposition of long amyloid-ß peptide (Aß) species, which are released from a C-terminal amyloid precursor protein fragment by γ-secretase. Mutations in its catalytic subunit presenilin-1 (PS1) increase the Aß42 to Aß40 ratio and are the major cause of familial AD (FAD). An opposing hypothesis states that loss of essential presenilin functions underlies the disease. A major argument for this hypothesis is the observation that the nearly inactive PS1 L435F mutant, paradoxically, causes FAD We now show that the very little Aß generated by PS1 L435F consists primarily of Aß43, a highly amyloidogenic species which was overlooked in previous studies of this mutant. We further demonstrate that the generation of Aß43 is not due to a trans-dominant effect of this mutant on WT presenilin. Furthermore, we found Aß43-containing plaques in brains of patients with this mutation. The aberrant generation of Aß43 by this particular mutant provides a direct objection against the presenilin hypothesis.

Important functional role of residue x of the presenilin GxGD protease active site motif for APP substrate cleavage specificity and substrate selectivity of γ-secretase.

γ-Secretase plays a central role in the generation of the Alzheimer disease-causing amyloid ß-peptide (Aß) from the ß-amyloid precursor protein (APP) and is thus a major Alzheimer's disease drug target. As several other γ-secretase substrates including Notch1 and CD44 have crucial signaling functions, an understanding of the mechanism of substrate recognition and cleavage is key for the development of APP selective γ-secretase-targeting drugs. The γ-secretase active site domain in its catalytic subunit presenilin (PS) 1 has been implicated in substrate recognition/docking and cleavage. Highly critical in this process is its GxGD active site motif, whose invariant glycine residues cannot be replaced without causing severe functional losses in substrate selection and/or cleavage efficiency. Here, we have investigated the contribution of the less well characterized residue x of the motif (L383 in PS1) to this function. Extensive mutational analysis showed that processing of APP was overall well-tolerated over a wide range of hydrophobic and hydrophilic mutations. Interestingly, however, most L383 mutants gave rise to reduced levels of Aß37-39 species, and several increased the pathogenic Aß42/43 species. Several of the Aß42/43 -increasing mutants severely impaired the cleavages of Notch1 and CD44 substrates, which were not affected by any other L383 mutation. Our data thus establish an important, but compared with the glycine residues of the motif, overall less critical functional role for L383. We suggest that L383 and the flanking glycine residues form a spatial arrangement in PS1 that is critical for docking and/or cleavage of different γ-secretase substrates.

Attenuated Abeta42 responses to low potency gamma-secretase modulators can be overcome for many pathogenic presenilin mutants by second-generation compounds.

Sequential processing of the ß-amyloid precursor protein by ß- and γ-secretase generates the amyloid ß-peptide (Aß), which is widely believed to play a causative role in Alzheimer disease. Selective lowering of the pathogenic 42-amino acid variant of Aß by γ-secretase modulators (GSMs) is a promising therapeutic strategy. Here we report that mutations in presenilin (PS), the catalytic subunit of γ-secretase, display differential responses to non-steroidal anti-inflammatory drug (NSAID)-type GSMs and more potent second-generation compounds. Although many pathogenic PS mutations resisted lowering of Aß(42) generation by the NSAID sulindac sulfide, the potent NSAID-like second-generation compound GSM-1 was capable of lowering Aß(42) for many but not all mutants. We further found that mutations at homologous positions in PS1 and PS2 can elicit differential Aß(42) responses to GSM-1, suggesting that a positive GSM-1 response depends on the spatial environment in γ-secretase. The aggressive pathogenic PS1 L166P mutation was one of the few pathogenic mutations that resisted GSM-1, and Leu-166 was identified as a critical residue with respect to the Aß(42)-lowering response of GSM-1. Finally, we found that GSM-1-responsive and -resistant PS mutants behave very similarly toward other potent second-generation compounds of different structural classes than GSM-1. Taken together, our data show that a positive Aß(42) response for PS mutants depends both on the particular mutation and the GSM used and that attenuated Aß(42) responses to low potency GSMs can be overcome for many PS mutants by second generation GSMs.