GLA Disease Concept v2.1

The Mechanistic Chain

This model illustrates the following cascade:

1. Viral/inflammatory trigger → TLR4 activation (VanElzakker et al., 2019; Che et al., 2025)
2. PKC → PI-PLC induction (Rostami-Afshari et al., 2025)
3. SMPDL3B cleavage → loss of lipid raft stability (Rostami-Afshari et al., 2025)
4. Endothelial dysfunction (Haffke et al., 2022)
5. Impaired perfusion → ischemic metabolism (Jones et al., 2010; Rutherford et al., 2016)
6. Ca²⁺ overload → ROS burst (Syed et al., 2025)
7. ROS → more PI-PLC → more SMPDL3B loss (Rostami-Afshari et al., 2025)
8. Kidney volume dysregulation amplifies ischemia (Farquhar et al., 2002; van Campen et al., 2018–2023)
9. Hepatic strain + FGF21 elevation (Hoel et al., 2021; Azimi et al., 2025)
10. Autonomic dysfunction further reduces perfusion (Issa et al., 2025; Christopoulos et al., 2025)

This creates interlocking feedback loops that sustain ME/CFS pathology.

1. The SMPDL3B Molecular Hub

SMPDL3B is a GPI-anchored membrane regulator that stabilizes lipid rafts, shapes TLR4 signaling, and maintains membrane fluidity under inflammatory and mechanical stress.

In ME/CFS, peripheral blood mononuclear cells show reduced membrane-bound SMPDL3B and increased soluble SMPDL3B, reflecting pathological cleavage of the protein from the cell surface (Rostami-Afshari et al., 2025).

This cleavage is mediated by PI-PLC (PLCXD1), an enzyme upregulated by TLR4 → PKC signaling, linking innate immunity directly to SMPDL3B dysregulation (Rostami-Afshari et al., 2025).

2. TLR4 Hyperreactivity → PI-PLC Activation

TLR4 activation produces:

• IL-6, TNF-α, IL-1β
• PKC stimulation
• downstream PI-PLC upregulation

In ME/CFS:

• IL-6 release after LPS stimulation is abnormally elevated
• PI-PLC protein is increased in plasma
• membrane SMPDL3B is reduced

(Rostami-Afshari et al., 2025; Che et al., 2025; Roerink et al., 2017). This creates a feed-forward inflammatory loop.

3. The SMPDL3B Cleavage Loop

Once PI-PLC is active:

1. It cleaves GPI-anchors.
2. SMPDL3B detaches from the cell membrane.
3. Loss of SMPDL3B removes TLR4 restraint.
4. TLR4 signaling intensifies.
5. PKC returns more stimulation to PI-PLC.

This forms a self-amplifying molecular circuit:

TLR4 → PKC → PI-PLC → SMPDL3B loss → TLR4 hyperreactivity (Rostami-Afshari et al., 2025; Nguyen et al., 2017; Blundell et al., 2015).

4. Two Distinct SMPDL3B Phenotypes

Based on the validated subtype data:

A. Shedding Phenotype (~50%, mostly women)

• High soluble SMPDL3B
• High PI-PLC
• Low membrane SMPDL3B
• High IL-6 response

B. Deficiency Phenotype (~25%, mostly men)

• Low soluble SMPDL3B
• Low PI-PLC
• Low membrane SMPDL3B
• Reduced functional SMPDL3B capacity

5. How SMPDL3B loss ties into the ME/CFS symptom network

Reduced membrane SMPDL3B results in:

• heightened inflammatory sensitivity
• oxidative stress
• impaired endothelial function
• microvascular constriction
• poor tissue perfusion under mild stress
• exaggerated post-exertional immune activity

(Rostami-Afshari et al., 2025; VanElzakker et al., 2019; Hardcastle et al., 2015). This molecular lesion is the entry point into downstream dysfunction.

1. Endothelial Dysfunction: The Primary Systems-Level Consequence

After SMPDL3B loss and TLR4 sensitization, the vascular endothelium becomes:

• inflamed
• vasoconstricted
• NO-depleted
• ROS-dominant
• metabolically inefficient

Multiple studies in ME/CFS and Long COVID document elevated ET-1 and dysregulated Ang-2, with important phase-dependent differences: Ang-2 is often increased during acute and early post-acute infection, whereas in Long COVID ME/CFS-like cohorts several months later Ang-2 may normalize or fall below control levels, particularly in the Haffke ME/CFS–Long COVID subgroup (Haffke et al., 2022; Flaskamp et al., 2022; Vassiliou et al., 2023).

2. Serum from ME/CFS/Long COVID induces endothelial injury

In vitro, endothelial cells exposed to ME/CFS or Long COVID serum show:

• increased permeability
• reduced tube formation
• oxidative stress
• impaired junctional proteins

(Flaskamp et al., 2022). This directly supports the microvascular limb of the model.

3. Microclots & RBC deformability across ME/CFS spectrum

Microclotting, fibrin amyloid formation, and impaired RBC deformability are observed in both Long COVID and ME/CFS, contributing to:

• reduced capillary flow
• oxygen extraction deficits
• increased local ischemia under mild load

(Nunes, Kell, & Pretorius, 2023).

4. Cerebral blood flow reductions

Tilt-table and Doppler studies consistently show 20–40% drops in cerebral blood flow in ME/CFS during orthostatic stress (van Campen et al., 2020, 2021, 2023; Christopoulos et al., 2025).

This supports the perfusion-failure component of your ischemia model.

1. Microvascular Under-Perfusion → Metabolic Stress

Under endothelial dysfunction, microvascular beds show:

• reduced oxygen delivery
• patchy perfusion
• impaired capillary recruitment

This drives anaerobic metabolism, intracellular acidosis, and metabolic strain (Jones et al., 2010; Rutherford et al., 2016).

2. ATP Depletion → Ca²⁺ Overload

When ATP is insufficient:

• SERCA and PMCA pumps fail
• Ca²⁺ accumulates in the cytosol and mitochondria
• mPTP opens
• ROS bursts occur

This matches PEM timing and the 24–72 h delayed crash window (Baraniuk, 2025; Syed et al., 2025).

3. ROS → PKC Activation → PI-PLC Upregulation

ROS activates PKC, which increases PI-PLC expression. This is documented directly in SMPDL3B-related work (Rostami-Afshari et al., 2025).

Thus: Ischemia → Ca²⁺ → ROS → PI-PLC → SMPDL3B cleavage → TLR4 hyperreactivity → more ischemia

This is a closed loop, explaining chronicity.

4. DAMP activation after exertion

Post-exertional release of:

• mtDNA
• succinate
• cardiolipin
• extracellular ATP

triggers innate sensing pathways including TLR4, amplifying inflammation (VanElzakker et al., 2019; Che et al., 2025).

This links PEM to the SMPDL3B–TLR4 axis.

5. Functional muscle evidence

Muscle studies show:

• abnormal pH recovery
• elevated resting sodium
• impaired oxidative metabolism
• reduced performance on repeat exercise tests

(Petter et al., 2022; Vermeulen et al., 2010; Franklin et al., 2022). This is direct physiological evidence of chronic ischemia-like behavior.

1. Podocyte SMPDL3B and mechanotransduction

SMPDL3B is a key podocyte regulator; its loss destabilizes:

• slit diaphragm signal transduction
• sodium handling
• bradykinin/RAAS balance

(Fornoni et al., 2011; kidney SMPDL3B literature summarized in Rostami-Afshari et al., 2025). This supports the “kidney–volume axis.”

2. Natriuresis & impaired volume retention

With podocyte dysfunction:

• sodium reabsorption signaling weakens
• aldosterone sensitivity may drop
• patients exhibit low blood volume and poor plasma expansion

(Farquhar et al., 2002; van Campen et al., 2018–2023).

3. Bradykinin dominance & medullary hypoxia

In reduced RAAS signaling:

• bradykinin rises
• renal vasodilation increases
• medullary O₂ tension falls
• ROS increases
• downstream PI-PLC activation worsens

This continues to fuel the vicious cycle. This sequence is inferred from renal physiology and SMPDL3B podocyte data, but has not yet been directly demonstrated in ME/CFS.

4. RAAS activation becomes ineffective

Despite low volume stimulating RAAS:

• angiotensin II signaling becomes blunted
• aldosterone may be insufficient
• plasma volume remains low
• orthostatic intolerance persists

(Jason et al., 2024; Issa et al., 2025).

5. Volume loss → systemic ischemia → SMPDL3B loss

Hypovolemia reduces perfusion further, feeding:

Low volume → hypoperfusion → ROS → PI-PLC → SMPDL3B cleavage → worse kidney function → lower volume

This matches clinical OI progression (Christopoulos et al., 2025).

1. Ischemia + Ca²⁺ overload as PEM engine

Muscle demonstrates:

• abnormal ATP recovery
• impaired aerobic function
• altered pH dynamics
• elevated intracellular sodium
• oxidative stress

(Petter et al., 2022; Jones et al., 2010; Rutherford et al., 2016).

2. Repeated exercise studies show pathological performance drops

Two-day CPET and repeated effort studies show:

• significant decline in function
• prolonged recovery
• biochemical abnormalities in CSF

(Vermeulen et al., 2010; Franklin et al., 2022; Baraniuk, 2025). This reinforces PEM as a vascular–metabolic injury, not deconditioning.

1. Cerebral hypoperfusion explains autonomic symptoms

Orthostatic intolerance in ME/CFS is linked to:

• 20–40% CBF reduction
• sympathetic overdrive
• impaired baroreflex integration

(van Campen et al., 2020, 2021, 2023; Christopoulos et al., 2025).

2. Autonomic dysfunction in large cohorts

Large-scale analyses (MCAM project) confirm:

• POTS
• neuropathic symptoms
• baroreflex abnormalities
• dysregulated HRV patterns

(Issa et al., 2025; Jason et al., 2024).

1. Hepatic metabolic stress

ME/CFS metabotyping shows:

• increased markers of hypoxia
• impaired β-oxidation
• abnormal amino acid signatures

(Hoel et al., 2021; Beentjes et al., 2025).

2. FGF21 elevation

FGF21 is elevated in subsets of ME and correlates with:

• symptom severity
• metabolic dysfunction
• neurocognitive patterns

(Azimi et al., 2025).

3. Liver involvement matches vascular–metabolic stress

FGF21 elevation is consistent with:

• mitochondrial strain
• sinusoidal endothelial dysfunction
• chronic oxidative stress

Similar patterns are seen in ME/CFS and Long COVID (Hoel et al., 2021; Low et al., 2023).

These annotations clarify which therapies are grounded in validated SMPDL3B biology vs. broader ME/CFS pathophysiology.

1. Targeting the SMPDL3B Axis

A. Shedding Phenotype: PI-PLC Inhibition

Individuals with this phenotype may theoretically benefit from DPP-4 inhibition (e.g., low dose saxagliptin), but this remains unproven and requires formal clinical trials.

Importantly, saxagliptin carries an elevated risk of heart-failure events in patients with pre-existing renal impairment, and should therefore be approached with caution.

B. Deficiency Phenotype: Metabolic Support

These individuals represent a non-shedding SMPDL3B-deficient state. Because PI-PLC is not elevated, inhibitors like saxagliptin do not address the root problem.

Instead, low dose metformin may improve:

• AMPK signaling
• mitochondrial stability
• cellular stress tolerance
• fatty acid oxidation

These changes help stabilize membrane environments indirectly (Hoel et al., 2021; Syed et al., 2025) and align with the metabolic fragility described in this subgroup.

2. Modulating TLR4 & Inflammatory Signaling

TLR4 hyperreactivity is a validated hallmark of ME immune alterations (Rostami-Afshari et al., 2025; Che et al., 2025; VanElzakker et al., 2019).

Therapeutic options that may dampen TLR4/innate pathways include:

• low-dose naltrexone (TLR4 antagonist)
• microdose lithium (anti-inflammatory modulation)
• curcumin analogues (TLR4/NF-κB inhibition)
• omega-3 fatty acids (resolvins → reduced IL-6/TNF-α)

These align with the IL-6 / TNF-α patterns documented across ME/CFS studies (Roerink et al., 2017; Hardcastle et al., 2015).

3. Restoring Endothelial & Microvascular Function

Given the consistent findings of:

• ET-1 elevation
• Ang-2 dysregulation with phase- and subgroup-dependent ↑ / ↓ patterns
• impaired microvascular dilatory capacity

(Haffke et al., 2022; Flaskamp et al., 2022; Vassiliou et al., 2023) therapeutic targets include:

• NO restoration strategies (L-arginine, BH4 support)
• mitochondrial antioxidants (CoQ10, PQQ, NAC)
• microcirculatory stabilizers
• antithrombotic/anti-microclot interventions (e.g., nattokinase, lumbrokinase)

These are consistent with microclotting and RBC deformability abnormalities (Nunes et al., 2023).

4. Correcting Blood Volume and RAAS–Bradykinin Imbalance

Low blood volume is documented across ME/CFS cohorts (Farquhar et al., 2002; van Campen et al., 2018–2023).

Interventions include:

• increased fluid + salt
• fludrocortisone (aldosterone mimic)
• midodrine (alpha-agonist)
• desmopressin (carefully, for volume restoration)

Because bradykinin-mediated vasodilation and renal hypoxia contribute to ROS-driven PI-PLC activity, stabilizing volume indirectly mitigates the ischemia loop.

5. Supporting the Hepatic / FGF21 Axis

FGF21 elevation in ME patients corresponds to metabolic stress, especially under exertion (Azimi et al., 2025).

Therapeutic considerations:

• mitochondrial support (riboflavin, CoQ10)
• reducing hepatic oxidative load
• stabilizing glucose and lipid metabolism
• gentle aerobic conditioning once stable

These align with metabotyping findings showing hypoxia-like metabolic signatures (Hoel et al., 2021; Beentjes et al., 2025).

6. Sympathetic & Parasympathetic Balancing

Autonomic dysfunction is universally observed in ME/CFS cohorts (Issa et al., 2025; Jason et al., 2024).

Targets include:

• vagal nerve stimulation (non-invasive)
• breathing training
• light physical maneuvers
• avoiding triggers of sympathetic overload

1. The Unified Model

The GLA/SMPDL3B disease model integrates validated findings:

• SMPDL3B cleavage from PI-PLC activation
• TLR4 hyperreactivity
• IL-6 and innate immune dysregulation
• endothelial dysfunction (ET-1, Ang-2, impaired NO)
• microvascular instability and ischemia
• low blood volume + RAAS/bradykinin imbalance
• mitochondrial and hepatic metabolic stress
• autonomic dysfunction

(Rostami-Afshari et al., 2025; Haffke et al., 2022; Hoel et al., 2021; Christopoulos et al., 2025; van Campen et al., 2020–2023).

2. Mechanistic Chain (Recap)

This model illustrates the following cascade:

1. Viral/inflammatory trigger → TLR4 activation (VanElzakker et al., 2019; Che et al., 2025)
2. PKC → PI-PLC induction (Rostami-Afshari et al., 2025)
3. SMPDL3B cleavage → loss of lipid raft stability (Rostami-Afshari et al., 2025)
4. Endothelial dysfunction (Haffke et al., 2022)
5. Impaired perfusion → ischemic metabolism (Jones et al., 2010; Rutherford et al., 2016)
6. Ca²⁺ overload → ROS burst (Syed et al., 2025)
7. ROS → more PI-PLC → more SMPDL3B loss (Rostami-Afshari et al., 2025)
8. Kidney volume dysregulation amplifies ischemia (Farquhar et al., 2002; van Campen et al., 2018–2023)
9. Hepatic strain + FGF21 elevation (Hoel et al., 2021; Azimi et al., 2025)
10. Autonomic dysfunction further reduces perfusion (Issa et al., 2025; Christopoulos et al., 2025)

This creates interlocking feedback loops that sustain ME/CFS pathology.

3. Clinical Pattern Explained

This unified model explains:

• PEM as a delayed ischemia–ROS–Ca²⁺ event
• persistent OI from poor cerebral and systemic perfusion
• muscle “poisoned” sensation from metabolic collapse
• sensory hypersensitivity from neuroinflammation
• hepatic discomfort during exertion or immune stress
• prolonged recovery windows
• heterogeneity (due to SMPDL3B shedding vs. deficiency subtypes)

4. Therapeutic Implications

• PI-PLC inhibition is appropriate only for the shedding phenotype (Rostami-Afshari et al., 2025).
• Metabolic support (metformin) aligns with the deficiency phenotype (Moreau, personal communication).
• Addressing endothelial and microvascular dysfunction is essential (Haffke et al., 2022; Flaskamp, 2022).
• Volume stabilization improves perfusion and reduces ischemia (van Campen et al., 2018–2023).
• Managing TLR4 hyperreactivity lowers inflammatory amplification (Che et al., 2025).
• Supporting mitochondrial and hepatic metabolism reduces crash severity (Hoel et al., 2021; Azimi et al., 2025).
• Autonomic modulation smooths the global physiological instability (Issa et al., 2025).