A biomarker of synapse loss, an early event in Alzheimer's disease (AD) pathophysiology that precedes neuronal death and symptom onset, would be a much-needed prognostic biomarker. With direct access to the brain interstitial fluid, the cerebrospinal fluid (CSF) is a potential source of synapse-derived proteins. In this study, we aimed to identify and validate novel CSF biomarkers of synapse loss in AD. Discovery: Combining shotgun proteomics of the CSF with an exhaustive search of the literature and public databases, we identified 251 synaptic proteins, from which we selected 22 for further study. Verification: Twelve proteins were discarded because of poor detection by Selected Reaction Monitoring (SRM). We confirmed the specific expression of 9 of the remaining proteins (Calsyntenin-1, GluR2, GluR4, Neurexin-2A, Neurexin-3A, Neuroligin-2, Syntaxin-1B, Thy-1, Vamp-2) at the human synapse using Array Tomography microscopy and biochemical fractionation methods. Exploration: Using SRM, we monitored these 9 synaptic proteins (20 peptides) in a cohort of CSF from cognitively normal controls and subjects in the pre-clinical and clinical AD stages (n ‫؍‬ 80). Compared with controls, peptides from 8 proteins were elevated 1.3 to 1.6-fold (p < 0.04) in prodromal AD patients. Validation: Elevated levels of a GluR4 peptide at the prodromal stage were replicated (1.3-fold, p ‫؍‬ 0.04) in an independent cohort (n ‫؍‬ 60). Moreover, 7 proteins were reduced at preclinical stage 1 (0.6 to 0.8fold, p < 0.04), a finding that was replicated (0.7 to 0.8fold, p < 0.05) for 6 proteins in a third cohort (n ‫؍‬ 38). In a cross-cohort meta-analysis, 6 synaptic proteins (Calsyntenin-1, GluR4, Neurexin-2A, Neurexin-3A, Syntaxin-1B and Thy-1) were reduced 0.8-fold (p < 0.05) in preclinical AD, changes that precede clinical symptoms and CSF markers of neurodegeneration. Therefore, these proteins could have clinical value for assessing disease progression, especially in preclinical stages of AD.

SummaryGenome editing and human induced pluripotent stem cells hold great promise for the development of isogenic disease models and the correction of disease-associated mutations for isogenic tissue therapy. CRISPR-Cas9 has emerged as a versatile and simple tool for engineering human cells for such purposes. However, the current protocols to derive genome-edited lines require the screening of a great number of clones to obtain one free of random integration or on-locus non-homologous end joining (NHEJ)-containing alleles. Here, we describe an efficient method to derive biallelic genome-edited populations by the use of fluorescent markers. We call this technique FACS-assisted CRISPR-Cas9 editing (FACE). FACE allows the derivation of correctly edited polyclones carrying a positive selection fluorescent module and the exclusion of non-edited, random integrations and on-target allele NHEJ-containing cells. We derived a set of isogenic lines containing Parkinson's-disease-associated mutations in α-synuclein and present their comparative phenotypes.

The juvenile form of neuronal ceroid Lipofuscinosis (JNCL) is the most common form within this group of rare lysosomal storage disorders, causing pediatric neurodegeneration. The genetic disorder, which is caused by recessive mutations affecting the CLN3 gene, features progressive vision loss, cognitive and motor decline and other psychiatric conditions, seizure episodes, leading to premature death. Animal models have traditionally aid the understanding of the disease mechanisms and pathology and are very relevant for biomarker research and therapeutic testing. Nevertheless, there is a need for establishing reliable and predictive human cellular models to study the disease. Since patient material, particularly from children, is scarce and difficult to obtain, we generated an engineered a CLN3-mutant isogenic human induced pluripotent stem cell (hiPSC) line carrying the c.1054C → T pathologic variant, using state of the art CRISPR/Cas9 technology. To prove the suitability of the isogenic pair to model JNCL, we screened for disease-specific phenotypes in non-neuronal two-dimensional cell culture models as well as in cerebral brain organoids. Our data demonstrates that the sole introduction of the pathogenic variant gives rise to classical hallmarks of JNCL in vitro. Additionally, we discovered an alteration of the splicing caused by this particular mutation. Next, we derived cerebral organoids and used them as a neurodevelopmental model to study the particular effects of the CLN3Q352X mutation during brain formation in the disease context. About half of the mutation -carrying cerebral organoids completely failed to develop normally. The other half, which escaped this severe defect were used for the analysis of more subtle alterations. In these escapers, whole-transcriptome analysis demonstrated early disease signatures, affecting pathways related to development, corticogenesis and synapses. Complementary metabolomics analysis confirmed decreased levels of cerebral tissue metabolites, some particularly relevant for synapse formation and neurotransmission, such as gamma-amino butyric acid (GABA). Our data suggests that a mutation in CLN3 severely affects brain development. Furthermore, before disease onset, disease -associated neurodevelopmental changes, particular concerning synapse formation and function, occur.