CSF Levels of Vgf correctly diagnose ALS and associates with clinical severity
Quantitative ELISA assay revealed that the decreased CSF levels of total full-length Vgf (P<0.05), correctly diagnosed ALS patients with 77% sensitivity and 87% specificity based on receiving operating characteristic (ROC) analysis (Figure A). Vgf CSF content decreased as a function of progression of muscle weakness characterized by an increasing number of affected muscle (segments) assessed by manual muscle testing (P<0.05) (Figure B).
Figure 1 Full length Vgf content in CSF in ALS. In A, full-length Vgf was assessed by quantitative ELISA assays; in B, Vgf content decreased as a function of progression of muscle weakness assessed by manual muscle testing revealing an increased number of affected (more ...)
Decreased Vgf content In CSF and serum precedes onset of ALS-type muscle weakness assessed by rotarod-assays
In our laboratory setting, G93A mutant SOD-1ALS mice develop muscle weakness by ~90 days of age (Figure A). The severity of motor impairment progresses to paralysis by ~130 days of age, followed by sacrifice.1
No detectable change in Vgf content in CSF and serum of G93A SOD-1 ALS mice was found in ~35 days old G93A SOD-1 ALS mice, relative to age-, gender-, and strain-matched wild-type littermates (Figure B,C).
Figure 2 Decreased Vgf content in the CSF and serum precedes ALS-type motor impairment assessed by rotarod assay. In A, ALS-type muscle weakness in mutant G93A SOD-1 as a function of clinical progression (age). In B,C, decreased Vgf levels in CSF and in serum (more ...)
Reduction in Vgf content in the CSF (F1,7793=4.913, P=0.0288 for age, F7,23660=2.131, P=0.0466 for Vgf content) and in the serum (F1,19840=5.4573, P=0.0345 for age, F7,34510 =3.945, P=0.0487 for Vgf content) in ~75 days old G93A SOD-1 mice was found to precede the onset of muscle weakness assessed by rotarod assay that normally occurs at ~90 days, relative to age-, gender-, and strain-matched wild-type littermates.
Serum and CSF content of Vgf continued to decrease up to ~130 days of age when G93A-SOD1 ALS mice are characterized by near complete paralysis, relative to gender-matched WT littermates (Figure B, C).
Vgf immunoreactive material in the lumbar (L3 to L5) spinal cord colocalizes with SMI-32 immunopositive motorneurons and decreases as a function of age progression in SOD-1 ALS mice
In the lumbar L3 to L5 region of spinal cord the distribution of Vgf immunoreactive material colocalized with SMI-32 immunoreactive spinal cord neurons (Figure A, panels 2-4). No detectable Vgf immunopositive signal was found to co-localize with NeuN immunoreactive neurons in the same spinal cord region (Figure B, panels 2-4), nor was there Vgf immunoreactive signal that co-localized with GFAP immunopositive astrocytes (data not shown).
Figure 3 Vgf immunoreactive material in the lumbar spinal cord co-localizes with SMI-32 immunopositive motorneurons and decreases as a function of age progression of SOD-1 ALS mice. In A, Vgf immunoreactive material is selectively localized within the nuclear (more ...)
Survey of Vgf content assessed immunocytochemically revealed that Vgf immunoreactive material in spinal cord motorneurons is already decreased in ~75 day old asymptomatic SOD-1 G93A-SOD1 ALS mice and continue to decrease as a function of progression of ALS-type muscle weakness up to ~130 days of age (Figure C)(F1,19840=14.28, P=0.0003 for age, F7,34510 =3.549, P=0.0018 for Vgf content), relative to age-, gender-, and strain-matched wild-type littermates.
The loss of Vgf immmunoreactive signal in SMI-32 spinal cord motorneurons in the L3-L5 region of spinal cord in ~130 days old mutant G93A-SOD1 ALS mice overlaps quantitatively with the loss in SMI-32 immunoreactive motorneurons assessed stereologically in the same tissue sections (Figure D), relative to age-, gender-, and strain-matched wild-type littermates.
Exogenous adenoviral Vgf expression protects G93A SOD1 mixed spinal cord neurons against excitotoxic injury
Total full-length mouse (m)vgf protein was expressed in ~8 days old primary mix mutant G93A SOD-1 spinal cord neuron cultures derived from E14 mouse embryos by infection with an adenoviral Vgf (Ad)-Vgf at 5 MOI.
Under this experimental condition, Ad-mVgf expression significantly increased intracellular Vgf and secreted full length Vgf protein (85 kDa) in the conditioned medium, relative to parallel Lac-Z infected cultures (5 MOI) (Figure , and Figure inset (not shown)), as assessed by western blot 48 hrs after infection. Next we explored the influence of exogenous Vgf expression in response to excitotoxic neuronal injury.
Figure 4 Exogenous adenoviral mVgf expression attenuates excitotoxicity in mixed cultures of spinal cord neurons. Neuron cultures were infected with Adeno (Ad)-Vgf or Ad-LacZ viral constructs at 5 MOI for 72 hrs, and then treated with AMPA (5 µM), NMDA (more ...)
We found treatment of control Lac-Z infected (5 MOI) primary mixed spinal cord neurons with glutamate receptor agonist AMPA (5 µM) or NMDA (20 µM), results in significant 30-40% loss in cell viability relative to untreated Lac-Z infected cultures (P<0.01; P<0.05, respectively), as assessed by LDH cell viability assay, 48 hr after treatment (Figure ).
We found that preventative expression of Ad-Vgf in primary mix mutant G93A SOD-1 spinal cord neuron cultures for 48 hrs resulted in significant protection against AMPA or NMDA mediated excitotoxic injury, as assessed by a neuronal viability assay 48 hr following drug treatment (Figure ).