Stem cell models of human synapse development and degeneration

Many brain disorders exhibit altered synapse formation in development or syn- apse loss with age. To understand the complexities of human synapse development and degeneration, scientists now engineer neurons and brain organoids from human-induced pluripotent stem cells (hIPSC). These hIPSC-derived brain models develop both excitatory and inhibitory synapses and functional synaptic activity. In this review, we address the ability of hIPSC-derived brain models to recapitulate synapse development and insights gained into the molecular mechanisms underlying synaptic alterations in neuronal disorders. We also discuss the potential for more accurate human brain models to advance our understanding of synapse development, degeneration, and therapeutic responses

Myosin IIA/IIB restrict adhesive and protrusive signaling to generate front–back polarity in migrating cells

Myosin IIA and IIB synergistically generate front–back polarity through their effects on actomyosin bundling formation and stability, and adhesion maturation, which are mediated by localized Rac GEF depletion

RhoGTPase Regulators Orchestrate Distinct Stages of Synaptic Development

Small RhoGTPases regulate changes in post-synaptic spine morphology and density that support learning and memory. They are also major targets of synaptic disorders, including Autism. Here we sought to determine whether upstream RhoGTPase regulators, including GEFs, GAPs, and GDIs, sculpt specific stages of synaptic development. The majority of examined molecules uniquely regulate either early spine precursor formation or later matura- tion. Specifically, an activator of actin polymerization, the Rac1 GEF β-PIX, drives spine pre- cursor formation, whereas both FRABIN, a Cdc42 GEF, and OLIGOPHRENIN-1, a RhoA GAP, regulate spine precursor elongation. However, in later development, a novel Rac1 GAP, ARHGAP23, and RhoGDIs inactivate actomyosin dynamics to stabilize mature synap- ses. Our observations demonstrate that specific combinations of RhoGTPase regulatory pro- teins temporally balance RhoGTPase activity during post-synaptic spine development

Non-muscle myosin II in disease: mechanisms and therapeutic opportunities

The actin motor protein non-muscle myosin II (NMII) acts as a master regulator of cell morphology, with a role in several essential cellular processes, including cell migration and post-synaptic dendritic spine plasticity in neurons. NMII also generates forces that alter biochemical signaling, by driving changes in interactions between actin-associated proteins that can ultimately regulate gene transcription. In addition to its roles in normal cellular physiology, NMII has recently emerged as a critical regulator of diverse, genetically complex diseases, including neuronal disorders, cancers and vascular disease. In the context of these disorders, NMII regulatory pathways can be directly mutated or indirectly altered by disease-causing mutations. NMII regulatory pathway genes are also increasingly found in disease-associated copy-number variants, particularly in neuronal disorders such as autism and schizophrenia. Furthermore, manipulation of NMII-mediated contractility regulates stem cell pluripotency and differentiation, thus highlighting the key role of NMII-based pharmaceuticals in the clinical success of stem cell therapies. In this Review, we discuss the emerging role of NMII activity and its regulation by kinases and microRNAs in the pathogenesis and prognosis of a diverse range of diseases, including neuronal disorders, cancer and vascular disease. We also address promising clinical applications and limitations of NMII-based inhibitors in the treatment of these diseases and the development of stem-cell-based therapies

Stem cell models of human synapse development and degeneration

Many brain disorders exhibit altered synapse formation in development or syn- apse loss with age. To understand the complexities of human synapse development and degeneration, scientists now engineer neurons and brain organoids from human-induced pluripotent stem cells (hIPSC). These hIPSC-derived brain models develop both excitatory and inhibitory synapses and functional synaptic activity. In this review, we address the ability of hIPSC-derived brain models to recapitulate synapse development and insights gained into the molecular mechanisms underlying synaptic alterations in neuronal disorders. We also discuss the potential for more accurate human brain models to advance our understanding of synapse development, degeneration, and therapeutic responses

Myosin IIB Activity and Phosphorylation Status Determines Dendritic Spine and Post-Synaptic Density Morphology

Dendritic spines in hippocampal neurons mature from a filopodia-like precursor into a mushroom-shape with an enlarged post-synaptic density (PSD) and serve as the primary post-synaptic location of the excitatory neurotransmission that underlies learning and memory. Using myosin II regulatory mutants, inhibitors, and knockdowns, we show that non-muscle myosin IIB (MIIB) activity determines where spines form and whether they persist as filopodia-like spine precursors or mature into a mushroom-shape. MIIB also determines PSD size, morphology, and placement in the spine. Local inactivation of MIIB leads to the formation of filopodia-like spine protrusions from the dendritic shaft. However, di-phosphorylation of the regulatory light chain on residues Thr18 and Ser19 by Rho kinase is required for spine maturation. Inhibition of MIIB activity or a mono-phosphomimetic mutant of RLC similarly prevented maturation even in the presence of NMDA receptor activation. Expression of an actin cross-linking, non-contractile mutant, MIIB R709C, showed that maturation into a mushroom-shape requires contractile activity. Loss of MIIB also leads to an elongated PSD morphology that is no longer restricted to the spine tip; whereas increased MIIB activity, specifically through RLC-T18, S19 di-phosphorylation, increases PSD area. These observations support a model whereby myosin II inactivation forms filopodia-like protrusions that only mature once NMDA receptor activation increases RLC di-phosphorylation to stimulate MIIB contractility, resulting i

Regulators of spine maturation are distinct from regulators of spine precursor formation.

<p><b>A)</b> Representative Images of GFP-expressing DIV-16 neurons transfected with the indicated shRNA targeting sequence for 48 hours. <b>B)</b> Regulators of spine precursor formation, OLIGOPHRENIN-1 (OPHN-1), β-PIX, and FRABIN, do not alter spine density later in synaptic development (DIV-16). Spine density is expressed as the percentage of the average control spine density. n = 44 control, 16 <i>Ophn-1</i> shRNA #1, 5 <i>Ophn-1</i> shRNA #2, 17 β<i>-pix</i> shRNA #1, 7 β<i>-pix</i> shRNA #2, 15 <i>Frabin</i> shRNA #1, 8 <i>Frabin</i> shRNA #2 neurons (Spine density was not significantly different from control as determined by t-test, except for β<i>-pix</i> shRNA #1 which was determined by Mann-Whitney Rank Sum Test). <b>C)</b> <i>Arhgap23</i> shRNAs significantly increase spine density later during synaptic development (DIV-16). n = 44 control (same as B), 22 <i>Arhgap23</i> shRNA #1, and 12 <i>Arhgap23</i> shRNA #2 neurons; p = 0.02 for Control vs <i>Arhgap23</i> shRNA #1 (Mann-Whitney Rank Sum Test), p = 0.002 for Control vs <i>Arhgap23</i> shRNA #2 (Mann-Whitney Rank Sum Test). <b>D)</b> Regulators of spine precursor formation, OLIGOPHRENIN-1 (OPHN-1), β-PIX, and FRABIN, do not alter spine length later in synaptic development (DIV-16) neurons. Cumulative distribution plot of spine length in DIV-16 primary rat hippocampal neurons co-expressing GFP and the indicated shRNA targeting sequence. Spine length is expressed as a percentage of the average control spine length. n = 3273 control, 651 <i>Ophn-1</i> shRNA #1, 130 <i>Ophn-1</i> shRNA #2, 729 β<i>-pix</i> shRNA #1, 449 β<i>-pix</i> shRNA #2, 556 <i>Frabin</i> shRNA #1, 688 <i>Frabin</i> shRNA #2 spines (Spine length was not significantly different from control as determined by Mann-Whitney Rank Sum test). <b>E)</b> <i>Arhgap23</i> and <i>Vav2</i> shRNAs significantly increase spine length later in neuronal development (DIV-16). n = 3273 control (same as D), 1207 <i>Arhgap23</i> shRNA #1, 1182 <i>Arhgap23</i> shRNA #2, 962 <i>Vav2</i> shRNA #1, and 551 <i>Vav2</i> shRNA #2 spines; p < 0.001 for Control vs <i>Arhgap23</i> shRNA #1 (Mann-Whitney Rank Sum Test), p < 0.001 for Control vs <i>Arhgap23</i> shRNA #2 (Mann-Whitney Rank Sum Test), p = 0.006 for Control vs <i>Vav2</i> shRNA #1 (Mann-Whitney Rank Sum Test), p < 0.001 for Control vs <i>Vav2</i> shRNA #2 (Mann-Whitney Rank Sum Test).</p

RhoGTPase Regulators Orchestrate Distinct Stages of Synaptic Development - Fig 6

<p><b>A & B) RhoGDIα and</b> γ <b>maintain spine maturation.</b> Quantification of Spine Length in GFP-expressing DIV16-27 neurons transfected with the indicated shRNA targeting sequence<b>s</b> or control neurons. Spine Length is normalized to the average control spine length for each neuronal culture. n = 1232 control spines, 1757 <i>Arhgdi-</i>α shRNA spines, 1305 <i>Arhgdi-</i>β shRNA spines, and 1526 <i>Arhgdi-</i>γ shRNA spines; p < 0.001 for control neurons vs either <i>Arhgdi-</i>α shRNA or <i>Arhgdi-</i>γ shRNA neurons, but is not statistically different from <i>Arhgdi-</i>β shRNA neurons (Mann-Whitney Rank Sum Test).</p

Rac drives spine precursor formation, while myosin-II and Cdc42 activity regulate spine length.

<p><b>A)</b> Rac1 photoactivation increases spine precursor formation. DIV14-21 primary rat hippocampal neurons expressing either photoactivatable Rac1 or as a positive control, constitutively activated Rac1 (‘Lit’ PA-Rac), were kept in dark (black bars) or exposed to room lighting for 10min (white bars). The resulting spine density is expressed as percent control unactivated PA-Rac-expressing neurons. n = 24 PA-Rac neurons kept in dark, 23 PA-Rac light-exposed neurons, 8 ‘lit’ PA-Rac neurons kept in dark, and 9 ‘lit’ PA-Rac light-exposed neurons; p = 0.047 PA-Rac dark vs light-acivated (t-test). <b>B)</b> Acute Rac1 photoactivation does not affect spine length, unlike constitutive Rac1 activity (‘Lit’ control). <b>C)</b> Representative images of neurons expressing either photoactivable Rac1 (PA-Rac1, top panel) or the constitutively active ‘lit’ Rac1 control (bottom panel) that were either kept in the dark (left panel) or exposed to room lighting for 10 min (light-activated, right panel). <b>D)</b> DIV-9/10 primary rat hippocampal neurons transfected with WT Raichu Cdc42 were treated with 50μM Blebbistatin for 1 hour or left untreated. FRET was calculated as the ratio of FRET signal to CFP donor signal. Blebbistatin treatment increases Cdc42 activity by ~7%. n = 50 spine precursors each for untreated and Blebbistatin-treated, p = 0.016 (t-test).</p