USP8 Deubiquitinates SHANK3 to Control Synapse Density and SHANK3 Activity-Dependent Protein Levels.

Mutations or altered protein levels of SHANK3 are implicated in neurodevelopmental disorders such as Phelan-McDermid syndrome, autism spectrum disorders, and schizophrenia (Guilmatre et al., 2014). Loss of SHANK3 in mouse models results in decreased synapse density and reduction in the levels of multiple synaptic proteins (Jiang and Ehlers, 2013). The family of SHANK scaffolding molecules are among the most heavily ubiquitinated proteins at the postsynaptic density. The ubiquitin-dependent proteasome degradation of SHANK is regulated by synaptic activity and may contribute to activity-dependent synaptic remodeling (Ehlers, 2003; Shin et al., 2012). However, the identity of the specific deubiquitinating enzymes and E3 ligases that regulate SHANK ubiquitination at synapses are unknown. Here we identify USP8/UBPY as a deubiquitinating enzyme that regulates SHANK3 and SHANK1 ubiquitination and protein levels. In primary rat neurons, USP8 enhances SHANK3 and SHANK1 protein levels via deubiquitination and increases dendritic spine density. Additionally, USP8 is essential for changes in SHANK3 protein levels following synaptic activity modulation. These data identify USP8 as a key modulator of SHANK3 downstream of synaptic activity.SIGNIFICANCE STATEMENT Precise regulation of the protein levels of the postsynaptic scaffolding protein SHANK3 is essential for proper neurodevelopment. Mutations of SHANK3 have been identified in Phelan-McDermid syndrome, autism spectrum disorders, and schizophrenia (Guilmatre et al., 2014). In this research, we identify USP8 as a key enzyme that regulates SHANK3 protein levels in neurons. USP8 acts to deubiquitinate SHANK3, which prevents its proteasomal-mediated degradation and enhances overall dendritic spine stability. In the future, the modulation of USP8 deubiquitinating activity could potentially be used to titrate the protein levels of SHANK3 to ameliorate disease.

Leukotriene A4 hydrolase inhibition improves age-related cognitive decline via modulation of synaptic function.

Leukotrienes, a class of inflammatory bioactive lipids, are well studied in the periphery, but less is known of their importance in the brain. We identified that the enzyme leukotriene A4 hydrolase (LTA4H) is expressed in healthy mouse neurons, and inhibition of LTA4H in aged mice improves hippocampal dependent memory. Single-cell nuclear RNA sequencing of hippocampal neurons after inhibition reveals major changes to genes important for synaptic organization, structure, and activity. We propose that LTA4H inhibition may act to improve cognition by directly inhibiting the enzymatic activity in neurons, leading to improved synaptic function. In addition, LTA4H plasma levels are increased in both aging and Alzheimer's disease and correlated with cognitive impairment. These results identify a role for LTA4H in the brain, and we propose that LTA4H inhibition may be a promising therapeutic strategy to treat cognitive decline in aging related diseases.

Noncanonical Activity of Tissue Inhibitor of Metalloproteinases 2 (TIMP2) Improves Cognition and Synapse Density in Aging.

Peripheral administration of tissue inhibitor of metalloproteinases 2 (TIMP2), a protein inhibitor of matrix metalloproteinases (MMPs), has previously been shown to have beneficial effects on cognition and neurons in aged mice. Here, to better understand the potential of recombinant TIMP2 proteins, an IgG4Fc fusion protein (TIMP2-hIgG4) was developed to extend the plasma half-life of TIMP2. Following one month of administration of TIMP2 or TIMP2-hIgG4 via intraperitoneal injections, 23-month-old male C57BL/6J mice showed improved hippocampal-dependent memory in a Y-maze, increased hippocampal cfos gene expression, and increased excitatory synapse density in the CA1 and dentate gyrus (DG) of the hippocampus. Thus, fusion to hIgG4 extended the half-life of TIMP2 while retaining the beneficial cognitive and neuronal effects. Moreover, it retained its ability to cross the blood-brain barrier. To deepen the mechanistic understanding of the beneficial function of TIMP2 on neuronal activity and cognition, a TIMP2 construct lacking MMP inhibitory activity, Ala-TIMP2, was generated, which provides steric hindrance that prevents inhibition of MMPs by the TIMP2 protein while still allowing MMP binding. A comprehensive assessment of the MMP inhibitory and binding capacity of these engineered proteins is outlined. Surprisingly, MMP inhibition by TIMP2 was not essential for its beneficial effects on cognition and neuronal function. These findings both confirm previously published research, expand on the potential mechanism for the beneficial effects of TIMP2, and provide important details for a therapeutic path forward for TIMP2 recombinant proteins in aging-related cognitive decline.

PCDH7 interacts with GluN1 and regulates dendritic spine morphology and synaptic function.

The N-terminal domain (NTD) of the GluN1 subunit (GluN1-NTD) is important for NMDA receptor structure and function, but the interacting proteins of the GluN1-NTD are not well understood. Starting with an unbiased screen of ~ 1,500 transmembrane proteins using the purified GluN1-NTD protein as a bait, we identify Protocadherin 7 (PCDH7) as a potential interacting protein. PCDH7 is highly expressed in the brain and has been linked to CNS disorders, including epilepsy. Using primary neurons and brain slice cultures, we find that overexpression and knockdown of PCDH7 induce opposing morphological changes of dendritic structures. We also find that PCDH7 overexpression reduces synaptic NMDA receptor currents. These data show that PCDH7 can regulate dendritic spine morphology and synaptic function, possibly via interaction with the GluN1 subunit.

CNS Neurons Deposit Laminin α5 to Stabilize Synapses.

Synapses in the developing brain are structurally dynamic but become stable by early adulthood. We demonstrate here that an α5-subunit-containing laminin stabilizes synapses during this developmental transition. Hippocampal neurons deposit laminin α5 at synapses during adolescence as connections stabilize. Disruption of laminin α5 in neurons causes dramatic fluctuations in dendritic spine head size that can be rescued by exogenous α5-containing laminin. Conditional deletion of laminin α5 in vivo increases dendritic spine size and leads to an age-dependent loss of synapses accompanied by behavioral defects. Remaining synapses have larger postsynaptic densities and enhanced neurotransmission. Finally, we provide evidence that laminin α5 acts through an integrin α3ß1-Abl2 kinase-p190RhoGAP signaling cascade and partners with laminin ß2 to regulate dendritic spine density and behavior. Together, our results identify laminin α5 as a stabilizer of dendritic spines and synapses in the brain and elucidate key cellular and molecular mechanisms by which it acts.