📘 GLA v2.3 — Updated Core Framework (2025)

1. Overview

GLA v2.3 unifies emerging biomarker findings from ME/CFS and Long COVID into a coherent, systems-level architecture describing how exertion intolerance and delayed crashes (PEM) arise from upstream regulatory impairments.

Two breakthroughs directly inform this update:

1) Long COVID EV-glycome abnormality (Pesqueira Sanchez et al., 2025)

Long COVID plasma contains:

• ↑ EV abundance
• ↑ high-mannose N-glycans on EV surfaces
• EV-associated inflammatory miRNAs removable by GNA (mannose-binding) resin

These findings establish a glycomic signature of unresolved ER/Golgi stress in a post-viral condition closely related to ME/CFS.

2) ME/CFS EV proteomic rigidity under exertion

ME/CFS cohorts demonstrate:

• Failure to remodel EV protein cargo after exertion (Giloteaux et al., 2024; Glass et al., 2025)
• Pathways affected include mitochondrial function, redox handling, immune signaling, and cellular stress responses

Together, these show EV biology as a core failure point in post-exertional adaptation.

GLA v2.3 integrates these findings with:

• ER-stress and hepatic metabolic bottlenecks (Hoel et al., 2021; Sun et al., 2019)
• SMPDL3B membrane/innate immune dysregulation (Rostami-Afshari et al., 2025)
• Endothelial instability and impaired perfusion signaling (Haffke et al., 2022)
• Autonomic and metabolic amplifier loops (van Campen et al., 2020–2023)

The result: a refined root architecture explaining how multiple micro-failures accumulate to produce PEM.

This version also integrates TUDCA as a mechanistically justified ER-chaperone intervention at the upstream regulatory layer.

2. Updated Root Structure (GLA v2.3)

2.1 EV Biology, ER Stress & Glycome Remodeling

Extracellular vesicles (EVs) carry inflammatory signals, mitochondrial stress markers, and regulatory RNAs. Their surface N-glycan patterns determine trafficking, immune interaction, and clearance.

Empirical evidence

Long COVID (Pesqueira Sanchez et al., 2025):

• ↑ EV counts (large & small)
• ↑ high-mannose N-glycans on EV surfaces
• GNA lectin resin selectively removes these EVs
• Removal depletes specific inflammatory miRNAs
• Predicted downstream pathway effects:
  ↓ JAK-STAT
  ↑ Estrogen / PI3K / VEGF

This represents a measurable EV-glycome abnormality associated with chronic post-viral illness.

ME/CFS (Giloteaux 2024; Glass 2025):

• EV proteomic remodeling fails after exertion
• Mitochondrial, redox, immune, and stress-response pathways do not normalize
• Indicates EV rigidity, but N-glycan structures have not yet been mapped

Acute COVID-19:

• High-mannose glycan enrichment
• ER/Golgi bottlenecks
• Glycosylation pathway dependence of viral entry

Working hypothesis (v2.3): EV-glycome insufficiency phenotype

Based on the convergence of EV rigidity (ME/CFS), EV-glycome abnormalities (Long COVID), and ER stress biology, ME/CFS may feature:

• ↑ high-mannose tone
• ↓ complex branching
• ↓ sialylation
• impaired post-exertional glycan remodeling

These traits are hypothesized and require direct measurement in ME/CFS EVs.

2.2 BA–GLA (Bile-Acid → Gut–Liver–Autonomic Axis)

The liver forms an upstream regulatory bottleneck. When ER and metabolic handling are impaired:

• FGF21 rises (Azimi et al., 2025)
• bile-acid signaling destabilizes (Sun et al., 2019)
• biochemical markers of hepatic strain appear (Beentjes et al., 2025)
• oxidative and redox demand increases (Fonseca et al., 2016)

These disturbances amplify:

• endothelial dysfunction
• autonomic instability
• metabolic collapse under exertion

BA–GLA is not “the cause” of ME/CFS, but the central integration point where upstream stress becomes systemic instability.

2.3 SMPDL3B, Innate Immunity & Membrane Stability

SMPDL3B regulates:

• membrane lipid organization
• TLR4 sensitivity
• microvesicle release
• cytoskeletal coherence

Evidence

• SMPDL3B is a validated ME/CFS biomarker (Rostami-Afshari et al., 2025)
• ME/CFS shows heightened innate immune activation (Che et al., 2025)
• Cytokine-pattern instability observed across multiple cohorts

GLA mechanistic phenotypes

(These are theoretical constructs, not clinical categories.)

• SMPDL3B-deficient phenotype:
  reduced membrane stability → exaggerated innate signaling
• SMPDL3B-shedding phenotype:
  excessive SMPDL3B loss → endothelial/cytoskeletal destabilization

These modulate how patients express downstream M1/M2/M3 amplifiers.

2.4 Vascular / Endothelial Amplifier (M2)

Characterized by:

• ET-1 elevation
• Ang-2 drift
• NO depletion
• microclots
• impaired capillary recruitment
• 20–40% cerebral blood-flow reduction on tilt

M2 is a perfusion-distribution failure, worsened by:

• hepatic strain
• EV inflammatory tone
• endothelial glycome vulnerability

2.5 Metabolic / Mitochondrial Amplifier (M1)

Ischemia + redox load produce:

• ATP depletion
• calcium overload
• ROS bursts
• abnormal lactate/pH dynamics
• slow phosphocreatine recovery

Reflects energy failure under load, intensified by M2 and BA–GLA upstream strain.

2.6 Autonomic / Volume Amplifier (M3)

Defined by:

• hypovolemia
• RAAS suppression
• sympathetic dominance
• baroreflex impairment
• POTS/OH
• reduced cerebral blood flow

Endothelial & EV-glycome abnormalities further limit autonomic adaptability.

3. PEM in GLA v2.3: A Failure of Integrated Remodeling

PEM is not caused by exertion itself but by the failure to return to baseline after exertion.

Supported contributors

• EV proteomic rigidity post-exertion
• innate immune overactivation
• microvascular and autonomic fragility

GLA integrative model of PEM

During recovery, multiple subsystems fail to remodel:

• EV proteome fails to normalize (empirical)
• EV-glycan remodeling likely impaired (inferred from Long COVID)
• endothelial/tight-junction instability
• hepatic metabolic bottlenecks
• TLR4-proximal innate amplification
• autonomic rebound failure

This creates the characteristic delayed crash, typically 12–48 hours post-exertion.

4. TUDCA in GLA v2.3

4.1 Mechanistic Rationale

TUDCA supports:

• ER chaperoning
• mitophagy/mitochondrial stability
• SIRT1-FXR activation
• improved glycan processing
• reduced ER Ca²⁺ leak

This directly targets the upstream defects identified by EV-glycome and BA–GLA findings.

4.2 Placement in the Model

TUDCA sits in the root layer, not downstream. It improves the regulatory environment for:

• endothelial function
• mitochondrial efficiency
• autonomic stability
• SMPDL3B membrane signaling

5. What GLA v2.3 Achieves

5.1 Clarifies the Root Dysfunction

• EV-glycome rigidity (shown in Long COVID)
• metabolic and hepatic strain (shown in ME/CFS)

5.2 Reframes Amplifier Types

M1/M2/M3 are not fixed “subtypes” but expressions of a shared upstream architecture, modulated by SMPDL3B biology, vascular tone, and hepatic load.

5.3 Establishes a Three-Level Treatment Logic

GLA v2.3 structures therapeutic strategy across three interconnected layers: (1) root regulatory stabilization, (2) midstream amplifier modulation, and (3) downstream rehabilitation. This layered architecture reflects how upstream cellular stress propagates into vascular, autonomic, and metabolic instability.

1) Root Regulatory Layer — correcting the upstream bottleneck

This layer targets ER stress, EV-glycome/proteome rigidity, hepatic metabolic load (BA–GLA), and SMPDL3B-related membrane/innate-immune vulnerability. The goal is to reduce systemic noise and create a low-friction metabolic and autonomic environment that prevents chronic activation of amplifier loops (M1, M2, M3).

Root-layer sequencing (autonomic-safe v2.3)

• Phase 0 — safety, autonomic settling, perfusion-risk avoidance
• Phase 1A — membrane/liver priming (PC first; others delayed in autonomic-fragile phenotypes)
• Phase 1B — ER stabilization
  TUDCA is introduced before any neuroactive BA–GLA amino acids, because choline, taurine, and glycine exert stronger acute effects on autonomic tone and can destabilize patients with M2 vascular dominance or SMPDL3B deficiency if used prematurely.
• Phase 1C — cautious introduction of choline → taurine → glycine
  (slow, phenotype-dependent ramping)
• Phase 2.5 — mitochondrial insulation (CoQ10 + riboflavin, niacinamide; ALA/ALCAR where appropriate)

This corrected ordering reflects the principle that upstream stabilization must occur before any intervention capable of shifting autonomic balance, particularly in vascular- or SMPDL3B-fragile phenotypes.

2) Midstream Layer — modulating amplifier expression (M1, M2, M3)

Once the root layer becomes more stable, the characteristic amplifier patterns of ME/CFS become more responsive to intervention. Midstream dysfunction includes:

• M2 (vascular): perfusion distribution failure, Ang-2/Tie2 drift, ET-1 elevation, microclots, NO depletion
• M1 (metabolic): impaired ATP/PCr recovery, ischemic ROS bursts, Ca²⁺ overload
• M3 (autonomic/volume): hypovolemia, RAAS suppression, baroreflex impairment, sympathetic dominance

Midstream interventions include endothelial repair, microvascular support, mitochondrial/redox optimization, cautious microclot modulation, and strategies that expand volume or stabilize autonomic tone.

Key principle: Improving the root layer reshapes amplifier expression. Patients frequently shift:
• M2 → M1 as vascular stability improves
• M3 → M2 as autonomic and volume status recover
• M1 → low-amplifier baseline as metabolic handling normalizes
Amplifier drift is a measurable indicator of upstream success.

3) Downstream Layer — rehabilitation and functional reintegration

Downstream work becomes viable only when root and midstream layers are stable. It includes:

• structured pacing with controlled reintroduction of exertion
• autonomic rehabilitation (graded autonomic loading, not graded aerobic exercise)
• perfusion-safe strengthening and conditioning
• sleep and circadian stabilization
• synchrony training across vascular, metabolic, and autonomic systems

Attempting rehabilitation prematurely typically reproduces PEM because the underlying regulatory architecture cannot yet remodel safely after exertion.

Overall, this three-layer structure provides a biologically grounded sequencing logic that links EV biology, ER stress, hepatic regulation, endothelial responsiveness, and autonomic behavior into a unified therapeutic framework.