UC San Diego

Fundamentally at the cellular level, Alzheimer's disease is a synaptic disease marked by loss of cortical glutamatergic synaptic density, and no widely used cellular model simultaneously preserves donor age and produces cortical neurons mature enough to interrogate disease-relevant synaptic biology. Direct conversion of adult human fibroblasts into induced neurons retains the donor's epigenetic age, but the neurons that emerge from standard three-week direct conversion sit in a hypo-mature state that does not fully elaborate cortical synaptic machinery reflected in the human adult brain. This thesis develops the NCA20+ direct-conversion framework, in which isoxazole-9 at 20 µM, a pro-neurogenesis small molecule, and a lipid-rich serum substitute, AlbuMax, are added into the previously established conversion protocol providing increased synaptic development and neuronal maturity in vitro. The resulting six-week protocol drives induced neurons through a two-phase Wnt-dependent cortical maturation trajectory. In Chapter 1 we show that the platform engages canonical Wnt signaling and proneural chromatin opening, followed by consolidation of non-canonical planar-cell-polarity Wnt signaling and cell-autonomous reconstitution of the astrocyte synaptogenic secretome, a critical and emerging component of Alzheimer's disease and aging pathology. Young and cognitively normal aged donor cells complete both phases and arrive at a mature cortical glutamatergic state; however, Alzheimer's disease donor cells fail to fully establish the canonical to non-canonical PCP transition, resulting in a marked deficit. The model, now capturing more detailed pathophysiology, revealed an Alzheimer's disease phenotype of 1) cell-fate instability consistent with synaptic deterioration, 2) incomplete chromatin consolidation through a pioneer-to-settler handoff deficit, 3) reduced cortical synaptic machinery transcription, and 4) reduced voltage-gated channel transcription. Whole-cell patch-clamp in young donor lines confirms the protocol yields functionally more mature neurons than standard conversion. Together, these demonstrate a potential Wnt signaling mechanism behind the cell fate instability in Alzheimer's disease. Having captured this pathology, we move on to Chapter 2 where we extend the platform in three directions: three-dimensional spheroid co-culture with hESC-derived astrocytes, live-cell endosomal transport dynamics at single-vesicle resolution, and transient partial epigenetic reprogramming and cellular rejuvenation through Oct4/Sox2/Klf4 expression. NCA20+ therefore enables mechanistic and interventional study in epigenetically age-matched, mature cortical neurons.