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Aberrant activity of cyclin-dependent kinase (Cdk5) has been implicated in various neurodegenerative diseases. This deleterious effect is mediated by pathological cleavage of the Cdk5 activator p35 into the truncated product p25, leading to prolonged Cdk5 activation and altered substrate specificity. Elevated p25 levels have been reported in humans and rodents with neurodegeneration, and the benefit of genetically blocking p25 production has been demonstrated previously in rodent and human neurodegenerative models. Here, we report a 12-amino-acid-long peptide fragment derived from Cdk5 (Cdk5i) that is considerably smaller than existing peptide inhibitors of Cdk5 (P5 and CIP) but shows high binding affinity toward the Cdk5/p25 complex, disrupts the interaction of Cdk5 with p25, and lowers Cdk5/p25 kinase activity. When tagged with a fluorophore (FITC) and the cell-penetrating transactivator of transcription (TAT) sequence, the Cdk5i-FT peptide exhibits cell- and brain-penetrant properties and confers protection against neurodegenerative phenotypes associated with Cdk5 hyperactivity in cell and mouse models of neurodegeneration, highlighting Cdk5i’s therapeutic potential.

APOE4 is the strongest genetic risk factor for Alzheimer’s disease (AD)(1–3). Yet, the effects of APOE4 on the human brain are not fully understood, limiting opportunities to develop targeted therapeutics for APOE4 and other AD risk factors(4–8). To gain more comprehensive insight into the impact of APOE4 on the human brain, we performed single-cell transcriptomics profiling of post-mortem human brains from APOE4-carriers compared to non-carriers. This revealed that APOE4 is associated with widespread gene expression changes across all cell types of the human brain. Consistent with APOE’s biological function(2–6), APOE4 significantly altered signaling pathways associated with cholesterol homeostasis and transport. Confirming these findings with histological and lipidomic analysis of the post-mortem human brain, iPSC-derived cells, and targeted-replacement mice, we further discovered that cholesterol is aberrantly deposited in oligodendrocytes, myelinating cells responsible for insulating and promoting electrical activity of neurons. We discovered altered cholesterol localization in the APOE4 brain coincides with reduced myelination. Pharmacologically facilitating cholesterol transport increases axonal myelination and improves learning and memory in APOE4 mice. Our study delivers a single-cell atlas detailing the transcriptional effects of APOE4 on the aged human brain and establishes a functional link between APOE4, cholesterol, myelination, and memory; opening paths to new therapeutic opportunities for AD.

In Alzheimer's disease, amyloid deposits along the brain vasculature lead to a condition known as cerebral amyloid angiopathy (CAA), which impairs blood-brain barrier (BBB) function and accelerates cognitive degeneration. Apolipoprotein (APOE4) is the strongest risk factor for CAA, yet the mechanisms underlying this genetic susceptibility are unknown. Here we developed an induced pluripotent stem cell-based three-dimensional model that recapitulates anatomical and physiological properties of the human BBB in vitro. Similarly to CAA, our in vitro BBB displayed significantly more amyloid accumulation in APOE4 compared to APOE3. Combinatorial experiments revealed that dysregulation of calcineurin-nuclear factor of activated T cells (NFAT) signaling and APOE in pericyte-like mural cells induces APOE4-associated CAA pathology. In the human brain, APOE and NFAT are selectively dysregulated in pericytes of APOE4 carriers, and inhibition of calcineurin-NFAT signaling reduces APOE4-associated CAA pathology in vitro and in vivo. Our study reveals the role of pericytes in APOE4-mediated CAA and highlights calcineurin-NFAT signaling as a therapeutic target in CAA and Alzheimer's disease.

Aberrant activity of cyclin-dependent kinase (Cdk5) has been implicated in various neurodegenerative diseases. This effect is mediated by pathological cleavage of the Cdk5 activator p35 to produce the truncated product p25, exhibiting increased stability and altered substrate specificity. The benefit of blocking p25 production has been demonstrated in various rodent and human neurodegenerative models. However, important Cdk5/p35 functions in the developing and adult brain have made it challenging to selectively target the detrimental effects of Cdk5/p25 while sparing the physiological functions of Cdk5/p35. Here, we report a 12-amino acid-long peptide fragment derived from Cdk5 (the Cdk5 inhibitory (Cdk5i) peptide) that shows a high binding affinity toward the Cdk5/p25 complex and can efficiently and selectively inhibit Cdk5/p25 kinase activity. Using cellular assays, mouse neurodegeneration models and human cerebral organoids generated from patient-derived iPSCs, we demonstrate beneficial effects of the Cdk5i peptide on various pathological phenotypes including gliosis, DNA damage, and Tau hyperphosphorylation.

Gait disabilities are common occurrences in patients and are treated with a variety of rehabilitation techniques. Current treatments include the use of a linear treadmill system to physically stimulate the patient. Present rehabilitation treadmills lack a visual component, which can help to increase functional changes in gait after training. Incorporating a virtual element has been shown to enhance gait rehabilitation outcomes [1]. The design described in this paper consists of three main components: a dual-belt, actuated treadmill, a virtual reality visual display and a motion capture system. The treadmill system emulates various slopes, curves and stairs while displaying a corresponding immersive environment. A dual-belt driven treadmill with four height actuators produces the various terrains. A live motion avatar, linked to a Qualisys motion capture system, is displayed on three screens to provide visual feedback and promote gait improvement. The immediate application of this system is to rehabilitate patients at the Veterans Affairs hospital and determine the effectiveness of visual and spatial feedback on gait recovery.