CNBY 645 Exam 3: Paper 1

Tumor microtubes

• Long cellular processes extended by glioma cells and riven by pathways involved in neurodevelopment and neuroplasticity

• Crucial for invasion and proliferation

• Connect single tumor cells to a functional communicating network

• Unknown involvement in the neuron-glioma interactions

Paracrine mechanisms

• Cell-to-cell communication in which a signal is released by a cell and acts on a neighboring cell

Autocrine mechanisms

• cell-to-cell communication in which a signal is released by a cell and acts on the same cell that produced it

Neuroglinin-3 neuronal protein (NLGN3)

• A neuronal cell adhesion molecule (CAM) that is a key regulator of synaptic function, found at both excitatory and inhibitory synpases

• Crucial for synapse development and forms connections between neurons

• Can be secreted by active neurons to promote tumor cell proliferation

NGS

• neurogliomal synapses

• Signal travels from neuron synapse to glioma cell

Electron microscopy (immuno-electron microscopy)

• A technique that uses antibodies labeled with electron-dense markers to visualize the precise location of molecules within a cell or tissue using a transmission electron microscope

What type of tumor studying? Cells, tissues, xenografts?

Studied glioblastoma cells and used xenografts of patient-derived human GB cell lines into mice

dSTORM

• Direct stochastic optical reconstruction microscopy

• A super-resolution imaging technique

• Achieves ~20nm resolution, far beyond standard light microscopy

• Works by turning fluorescent molecules “on and off” randomly and reconstructing their precise location

• In the paper, dSTORM was used to visualize synaptic proteins at glioma tumor microtubules (TMs) and confirm the presence of bona fide postsynaptic structures

HOMER1, HOMER2, and HOMER3

Postsynaptic density scaffold proteins found at excitatory (glutamatergic) synapses

Role: 

• Organize and cluster receptors (like AMPARs and mGluRs)

• Anchor them to postsynaptic density

• In the paper: HOMER1/2/3 served as markers of postsynaptic specialization on glioma TMs

  • Detection of HOMER proteins in glioma TMs confirmed that glioma cells form postsynaptic-like sites at neuron-glioma synapses

VGLUT1 (vesicular glutamate transporter 1)

• A presynaptic protein that loads glutamate into synaptic vesicles

• Marker of glutamatergic presynaptic boutons (where glutamate is released)

• In the paper: VGLUT1 puncta were found were found aligned with glioma postsynaptic AMPARs (GluR1 subunits), proving that the input gliomas receive is glutamatergic

AMPAR

• A glutamate receptor that mediates fast excitatory neurotransmisiion

• Plays a crucial role in learning and memory by enabling synaptic plasticity

Glutamate

• An excitatory transmitter in the CNS

5-ALA

• 5-aminolevulinic acid

• Is a fluorescent dye that causes tumor cells to glow and helps distinguish between cancerous and healthy tissue

NMDAR

A glutamate receptor that opens a channel allowing calcium influx, triggering a cascade of events that result in long-term changes in synaptic strength

Slice culture

A technique that uses a thin slice of tissue to analyze for research purposes

Co-culture

A technique in which two or more different cell types are grown together in the same culture to study their interactions 

Patch clamp

• Uses a glass micropipette filled with electrolyte solution to record the electrical currents and voltage across a small patch of cell membrane or across a cell

• Reveals insights into neuronal activity, cellular function, and the effects of drugs

sEPSCs

• spontaneous excitatory post-synaptic currents

• Small, rapid changes in a neuron’s membrane potential that occur spontaneously and increase the likelihood of the neuron firing an action potential

• Arises from random, localized release of neurotransmitters

• Often mediated by non-NMDA receptors, which cause an influx of positive ions and depolarize post-synaptic cell

• Helps explain fundamental synaptic transmission, the role of different receptors and how neurons respond to normal activity

CNQX

• Cyanquixaline

• A competitive AMPAR antagonist

TTX

• Tetrodotoxin

• A powerful neurotoxin (often found in pufferfish) that blocks voltage-gated sodium channels in nerve and muscle cells 

NASPM

• 1-napthylacetyl spermine

• Acts as a selective antagonist that inhibits calcium-permeable AMPPA receptors

Spontaneous slow inward currents (SICs)

• Excitatory events in neurons caused by the release of glutamate from astrocytes that activates extrasynaptic NMDARs

• Have slow kinetics (rise/decay time) and large amplitude

• Thought to synchronize small groups of neurons and regulate synaptic plasticity

• Blocked by NMDA receptor antagonists and illustrate a NMDA receptor dependency

Glioma calcium currents

• These are changes in electrical charge inside glioma cells caused by calcium flowing through ion channels, particularly calcium-permeable AMPA receptors (AMPARs)

In glioma, calcium currents:

• Signal through tumor cell networks (via tumor microtubes)

• Promote cell invasion and proliferation

• Are triggered by neuronal activity at neurogliomal synapses (NGS) 

Multiphoton Laser Scanning Microscopy (MPLSM)

• A powerful live imaging technique that uses two-photon or multiphoton excitation

• Allows researchers to look deep into the living brain of mice with minimal damage

• In this study: used to record calcium activity in glioma cells in real time through the cranial window

Cranial window

• A surgical procedure where part of the skull is removed and replaced with a transparent cover

• Enables repeated in vivo imaging of the brain (using MPLSM)

• In this study: it was used to visualize glioma cells in live mice, track their calcium signals, and measure invasion velocity

Channelrhodopsin-2 (ChR2)

• A light gated ion channel from algae used in optogenetics

• When expressed in neurons, it allows researchers to control neuronal activity with blue light

• In this paper: stimulating ChR2-expressing neurons with blue light increased neuronal firing —> drove synchronized calcium signals in glioma cells

Epileptiform activity- gabazine

• gabazine is a drug that blocks GABA-A receptors, which normally mediate inhibitory (calming) signals in the brain

• Blocking them induces hyperactive, seizure-like activity (epileptiform activity)

• In this paper: Gabazine was used to mimic epileptic conditions in vivo —> this increased glioma calcium currents and enhanced tumor network activity

Anesthesia

• Isoflurane impacts on calcium networks: isoflurane is commonly used anesthetic

• In glioma-bearing mice:

  • Isoflurane suppressed calcium transients and reduced coordinated glioma network activity

  • Also slowed glioma cell invasion in vivo

  • Shows how brain activity levels directly impact glioma cell behavior

Invasion velocity- repetitive in vivo imaging

• Researchers tracked how fast glioma cells moved (their invasion velocity) using time-lapse in vivo imaging through the cranial window

• Glioma cells with functional AMPARs and neuronal input invaded faster

• Anesthesia (isoflurane) or genetic AMPAR blockade (GluA2-DN) reduced invasion velocity

GluA2-DN-GFP

• Dominant-negative (DN) mutant version of GluA2 (tagged with GFP for visualization) was expressed in glioma cells

• This blocks AMPAR signaling in those cells

• Result: Glioma cells with GluA2-DN-GFP showed slower invasion and reduced proliferation compared to control glioma cells

Optogenetic construct

• A genetic tool that makes glioma or neuron cells light-sensitive (e.g. by expressing ChR2)

• When illuminated, neurons fire —> this increases glutamatergic synaptic activity onto glioma cells

• Used to prove that neuronal activity directly stimulates glioma growth and invasion

Perampanel

• A clinically approved antiepileptic drug that selectively blocks AMPA receptors

• In the study:

  • Chronic perampanel treatment in mice slowed flioma growth

  • Reduced glioma cell density increases over time

• Suggests repurposing perampanel as glioma therapy

Main goal of study

• Hopes to find that neurons and glioma cells form direct, bone fide chemical synapses called neurogliomal synapses (NGS)

Figure 1

Goal: Show that glioma cells physically form bona fide synapses with neurons

Methods: Immuno-EM images

Key takeaway: NGS are consistently formed in incurable human gliomas and mouse models thereof but not in less malignant primary brain tumors, suggesting a specific contribution to the malignant features of astrocytic gliomas, including GB

Figure 2

Goal: Show glioma cells express synaptic proteins and AMPA receptors

Methods: dSTORM micoscopy, confocal imaging, sRNAseq, EM

Key numbers: Across xenograft models and resected human GB material >80% of AMPAR signals on glioma TMs colocalized with presynaptic signals

Key takeaway: Glioma cells molecularly organize like neurons at NGS with AMPAR-rich synaptic contacts, especially in TM-connected subpopulations

Figure 3

Goal: prove that NGS are functional synapses

Methods: patch clamp, sEPSCs

Key takeaway: all of this data combined with knowledge from previous studies indicates that there is a relevant electrophysiologically heterogeneous functional input of NGS on GB cells

Figure 4

Goal: determine what are the effects in the glioma cells of the NGS and currents

Key takeaway: Calcium can enter GB cells via AMPARs and possible additional conductances and that neuronal activity can cause time-locked and clinically relevant calcium signals in GB cell networks

Figure 5

Goal: analyze the consequences of NGS and its effect on tumor growth

Shows that isoflurane treatment visibly slows invasion of tumor microtube-bearing GB cells

Conclusion

• Reframes gliomas as active participants in neural circuits

• Gliomas hijack CNS synaptic mechanisms by forming function NGS, providing excitatory AMPAR-mediated input to glioma cells; this drives calcium signaling within tumor microbe-connected networks and drives invasion/proliferation

• Suggesting seizures in glioma patients may not just be symptom of tumor growth but also actively fuel tumor progression by increasing neuronal activity

• Therapeutic: drugs that block synaptic input (like perampanel) may be repurposed for glioma treatment