Fig 2: Group-wise comparison of z-scores from age- and sex-corrected total levels of brevican (A,B) and neurocan (C,D) as measured via ELISAs in CSF (A,C) and serum (B,D) samples (CSF BCAN: Ctr. n = 26, ALS n = 16, Epi n = 25, CSVD n = 22; CSF NCAN: Ctr. n = 26, ALS n = 15, Epi n = 24, CSVD n = 22; Serum BCAN: Ctr. n = 23, ALS n = 10, Epi n = 24, CSVD n = 22; Serum NCAN: Ctr. n = 26, ALS n = 10, Epi n = 26, CSVD n = 22). No statistical significances were detected between groups. (E) The concentrations of total brevican and total neurocan in the CSF correlate with each other (rρ = 0.6211, p < 0.0001). CSF, cerebrospinal fluid; Ctrl, control; ALS, amyotrophic lateral sclerosis; Epi, epilepsy; CSVD, cerebral small vessel disease.

Fig 3: Ncan and synaptic markers in VCN. Changes in Ncan expression were accompanied by loss of glutamatergic synaptic boutons (a–c, arrows). Inset shows double labeling of Ncan and the neuronal marker HuC/HuD in VCN by POD3, with Ncan(+) PNNs exclusively covering neurons. GABAergic boutons (arrows) gained in staining intensity by POD3 (e) and POD7 (f). Most GABAergic boutons seemed to be embedded in Ncan(+) PNNs (arrowheads) by POD3 (e). Gap43(+) profiles (arrows) were often found close but outside the Ncan(+) PNNs by POD3 (h), but in contact to cell bodies by POD7 (i). Neuronal cell bodies are indicated by asterisks. Scale bar for a–i: 20 µm; scale bar for inset: 20 µm. In the quantification of vGluT1 (j), staining ratio dropped by POD3 and remained low. Ratio of Gad65 (k) rose by POD3

Fig 4: Interaction of perineuronal net components and tau protein. The interaction of several perineuronal net (PN) components and tau protein was investigated by combined co-immunoprecipitaion and Western blot analysis. (A) Verification of the target structure extraction (aggrecan (ACAN): AB1031 (Merck Millipore), at ~450 kDa; brevican (BCAN): clone 2 (BD Bioscience), at ~ 145 kDa; neurocan (NCAN): AF5800 (R&D Systems), at ~150 kDa; HAPLN1: AF2608 (R&D Systems), at ~40 kDa) by Dynabead-antibody-complex. Target structure specific bands were marked by an asterisk. The prominent signal about 55 kDa is attributed to the IgG heavy chain of the capture antibodies. (B) The formation of a PN-molecule-tau-complex was determined by total tau antibody (anti-pan tau (Dako)). A prominent tau-specific signal at ~65 kDa was detected when using C57BL6 brain homogenates for co-immunoprecipitation. When using brain homogenates of Mapt KO mice, no tau-specific signal was detected. The band pattern below 55 kDa in C57BL6 and Mapt KO mice is attributed to the IgG heavy and light chain of the applied capture antibodies. For illustration of exemplary blot lanes, brightness and contrast were adapted.

Fig 5: Ncan staining in dorsal cochlear nucleus (DCN), lateral superior olive (LSO), and medial geniculate body (MGB) by POD3. In DCN (a, b), a rise of Ncan expression pattern was reminiscent to that in VCN but weaker. In LSO (c, d, outlined by dashed line), we found scattered Ncan(+) PNNs (arrowheads) in controls and at all survival times, with no differences between hemispheres. In MGB (e, f) Ncan staining was absent from either side in any experimental group. Neuronal cell bodies are indicated by asterisk. MGBv, ventral part of the medial geniculate body; MGBd, dorsal part of the medial geniculate body; MGBm, medial part of the medial geniculate body; MSO, medial superior olive. Scale bars for a–d: 200 µm; scale bars for insets: 20 µm