The pathophysiological changes associated with neurodegenerative diseases (NDDs) begin decades before the emergence of clinical symptoms. Understanding the early mechanisms associated with NDDs pathology is, therefore, important for identifying disease-modifying therapeutic targets. Recent genetic and biochemical analysis has confirmed that the abnormal accumulation of naturally unfolded proteins in the brain is central to several NDDs. These "cerebral proteopathies" include dementia with Lewy bodies (DLB), Alzheimer's disease (AD), Parkinson's disease (PD) and a variety of other disorders. AD, the most common form of dementia, is characterized by accumulation of intraneuronal deposition of the hyperphosphorylated tau protein (neurofibrillary tangles) and extracellular aggregates of β-amyloid (amyloid plaques). However, amyloid accumulation often also occurs in patients with DLB and in some patients with PD with dementia [1]. At the same time more than 50% of AD cases exhibit abundant brain accumulation of α-synuclein (α-syn) positive Lewy bodies (LBs) [2],[3]. On the other hand, α-syn accumulates in PD and is considered as a potential biomarker of this disorder.

α-Syn (α-syn) is ubiquitously expressed in brain and is highly enriched in presynaptic nerve terminals, where its main physiological function is the regulation of synaptic levels of monoamine neurotransmitters through modulation of vesicular release. The results of analysis of total α-syn in peripheral blood, plasma and cerebrospinal fluid (CSF) do not confirm that it can be used as a reliable biomarker or predictor of disease progression. However, post-translationally modified forms of α-syn and in particular Ser¹²⁹ phosphorylated isoform (phospho-α-syn) [4] might have potential value as a diagnostic tool [5],[6].

Quantification of post-translationally modified forms of these proteins elicits more hope to find a new biomarker useful for diagnostic purposes. The finding and characterization of dependable novel biomarkers is still highly unmet and much needed. Initial findings of new biomarkers using post-mortem brain samples may lead to their subsequent testing with biological fluids, i.e. serum or cerebrospinal fluid and become a basis for the development of new diagnostic tools.

Another member of the synuclein family - γ-synuclein (γ-syn) is also aggregation-prone protein which forms intracellular inclusions. γ-Syn is a component of atypical inclusion bodies in human NDDs [7]-[11]. Furthermore, elevated expression of γ-syn causes selective damage and loss of discrete populations of neurons and their axons [9],[12]. Importantly, polymorphisms in the γ-syn locus have been associated with human diffuse Lewy body disease [13]. Formation of heteromeric complexes containing α- and γ-syn was demonstrated [14]. Our recent results indicate that γ-syn is easily oxidized at Methionine-38 (Met-38) forming oxidized-γ-syn (oxi-γ-syn) and after oxidation it can induce aggregation of α-syn [15]. Moreover, γ-syn can be transmitted between cells [15] and presumably to spread pathology as was shown for α-syn [16]-[18]. These findings raise a possibility that oxidation and/or aggregation of this protein might be involved in the pathogenesis of some NDDs. This prompted us to investigate if γ-syn pathology is present in patients with AD with LB, DLB and PD.

Naturally unfolded proteins, including synucleins, are often post-translationally modified which may serve as a signal for their sequestration in inclusion bodies [4],[19],[20]. Previously we have found that γ-syn can be easily oxidized and form aggregates and inclusions in cell cultures [15]. Here we used antibodies specific to unmodified and post-translationally modified α- and γ-syns to reveal immunopathology in brain tissues of patients with NDDs. In particular, we used antibodies to pSer¹²⁹-α-syn, α-syn specific antibody raised against LB and antibody to oxi-Met³⁸-γ-syn. We also identified several types of oxi-γ-syn positive astrocytes with different morphology and examined their immunohistochemical phenotypes. Finding of new astrocytes markers which could distinguish different types of astrocytes is important for better understanding of their role in healthy brain and in NDDs.

Brain specimens of the amygdala and the substantia nigra were obtained from the brain bank of the University of Kansas Medical Center Alzheimer's Disease Center. Tissue was requested from pathologically-defined cases with the presence of Lewy bodies and included individuals with AD-related changes, PD, and DLB. A primary diagnosis was defined based on the burden of pathology and neuropathological characteristics.

Paraffin sections were used to retrieve two formalin-fixed, paraffin-embedded blocks, one for the amygdala and the other for the substantia nigra. From each of these paraffin blocks, 7 μm sections were cut and mounted on microscope slides. The formalin-fixed paraffin-embedded brain sections were deparaffinized by standard method. Heat-mediated antigen retrieval procedure using 10 mM sodium citrate buffer, pH-6.0 was applied. Permeabilization was done in PBS containing 0.2% Triton X-100 for 45 min at RT. Blocking was carried out overnight at +4°C in 10% normal horse serum containing 0.05% Triton-X-100.

PTMs have emerged as important determinants of protein physiological and pathological functions. Several PTMs are enriched within LB and exist at higher levels in α-synucleinopathy brains. Synuclein's PTMs interfere with its aggregation propensity and cellular signaling [19]–[22]. These findings suggest that PTMs of synucleins may signal a shift to a toxic gain of function and/or sequestration into insoluble structures. Here we have shown that phospho-α-syn and oxi-γ-syn are colocalized in LB in amygdala and substantia nigra of DLB, PD and some AD patients. The results of Western blotting show the absence of cross reactivity between antibodies we used for identification of α- and γ-syn. We also revealed another type of γ-syn immunopositive staining of astrocytes which are present both in DLB patients and in control brain samples. The presence of oxi-γ-syn in intracellular deposits may have an important role in the process of inclusion formation, since oxi-γ-syn induces protein aggregation [15] and thus may initiate protein deposition.

Interestingly, intracellular inclusions similar to doughnut-like LB shown on Figure 2B-D have been described recently in DLB patients using antibodies to α-syn and caspase-cleaved TDP-43 [23]. Both markers are uniformly distributed in these structures in PD cases, but peripherally distributed in patients with DLB.

In contrast to formation of synuclein inclusions in neurons, we did not find similar inclusions and deposits in astrocytes, although glial accumulation of α-syn was described previously, especially at the step of initiation of NDDs [24],[25]. The role of neuron-glia interaction in the etiology of NDDs is an important area of current investigations due to the involvement of naturally unfolded proteins in prion-like transmission of pathology [16]–[18],[24]. The results presented here are in agreement with previous observations of aberrant γ-syn accumulation in the optic nerve astrocytes and spheroids which is not accompanied by the formation of inclusion bodies or deposits [10],[11]

We identified several types of oxi-γ-syn positive astrocytes with different morphology and examined their immunohistochemical phenotypes. Some of them are compact with short processes (Figure 4A and D) looking similar to protoplasmic astrocytes [26],[27], others have longer processes (Figure 4D and G) which may end on a blood vessel (Figure 5C). Some of the oxi-γ-syn positive astrocytes display mixed morphological and immunocytochemical phenotypes between protoplasmic and fibrous astrocytes (Figure 4A and C). The quantity of oxi-γ-syn positive astrocytes differs significantly in patients with different NDDs and controls. They are most frequent in patients with PD (Table 1). Astrocytic α-syn is considered an important component of the pathology [28],[29], however the role of γ-syn and post-translationally modified forms of synucleins in neurodegeneration is not completely understood.

The results presented here might be important for better understanding of mechanisms initiating the formation of protein deposits in NDDs. Findings from several recent studies have suggested that aggregation-prone proteins, such as tau, α-syn, polyglutamine-containing proteins, and amyloid-β, can enhance each other's aggregation [30]–[35]. In particular, β-amyloid augments α-syn accumulation [33],[34], Tau increases α-syn aggregation [31],[32] while synergistic interactions between β-amyloid, tau, and α-syn might accelerate neuropathology [33]. Importantly, mechanism of hybrid oligomer formation plays an essential role in the pathogenesis of combined AD and PD [30].

A role of γ-syn and its post-translationally modified forms in NDDs are not investigated in such details as of α-syn and other naturally unfolded proteins. However, neuronal accumulation of aberrant γ-syn in NDDs suggests that γ-syn plays an important, yet not completely understood role in these disorders. Furthermore, its presence in cerebrospinal fluid and elevation in aged subjects with neurodegenerative and vascular changes suggest that γ-syn can be used as a biomarker of neurodegeneration, gliosis in dementia with Lewy bodies and other NDDs [36].

Recent studies have demonstrated that α- and γ-syn interact with each other and these interactions increase the propensity of synucleins to aggregate [15],[37]. Co-immunoprecipitation experiments showed heteromeric protein: protein complexes that included both α- and γ-syns [14],[15]. Thus, antibody to oxi-γ-syn can be used as a new biomarker for NDDs both individually and in combination to antibodies to other markers of neurodegeneration. The presence of both α-syn and γ-syn in brain deposits in several NDDs may suggest a common element in the mechanism leading to these disorders.

Astrocytes exert many essential and complex functions and react to all forms of CNS insults, including NDDs with a response referred to as reactive astrogliosis. Finding of new astrocytes markers which could differentiate various types of astrocytes is important for better understanding of their role in healthy brain and in NDDs.

The results presented here confirm previous findings that accumulation of γ-syn may occur in human brain. Lesions immunopositive for both α-syn and γ-syn, as well oxi-γ-syn positive astrocytes with different morphology are described.

Conceived and designed the experiments: IS AS KLN.Performed the experiments: IS. Analyzed the data: IS, JB, AS, KLN. Contributed reagents/materials/analysis tools: IS, KLN, AS. Wrote the paper: AS, JB, KLN, IS. All authors read and approved the final manuscript.