All antibodies targeting intracellular antigens are paraneoplastic except anti-GAD65 antibodies, anti-Homer3, and anti-AK5. The detection of these antibodies confirms the immune-mediated origin of the neurological disorder and is helpful in tumor search. The following immunohistochemical staining patterns can be observed:

Hu antibodies (type 1 anti-neuronal nuclear autoantibodies, ANNA1) and Ri antibodies (type 2 anti-neuronal nuclear antibodies, ANNA2) label nuclei and cytoplasm of neurons in cerebrum, cerebellum, and brain stem; nucleoli are spared (Fig. 12.1a). In the peripheral nervous system, Hu-antibodies stain the myenteric plexus, while Ri-antibodies do not. Both antibodies bind to neuron specific RNA-binding proteins with wide distribution in the nervous system (Graus et al. 2001; Pittock et al. 2003). Anti–Yo antibodies (PCA1) label the cytoplasm of Purkinje cells and some stellate and basket cells in the molecular layer of the cerebellum (Fig. 12.1b). The antibodies recognize the cerebellar degeneration–related protein 2 (CDR-2), a Purkinje cell protein that is involved in signal transduction and gene transcription. Anti–CV2 antibodies (CRMP5) label oligodendrocytes in the white matter of cerebellum, brainstem, and spinal cord (Fig. 12.1c). The antibodies target a cytoplasmic protein of the collapsin response mediator protein family with a potential role in synaptic events (Honnorat et al. 1996). Anti–amphiphysin antibodies show a synaptic staining of the molecular and granular cell layer of the cerebellar cortex, and midbrain, the Purkinje cells are unstained (Pittock et al. 2005). Amphiphysin is a synaptic vesicle protein that is important for vesicle membrane recycling after depolarization. Anti–Ma1/2 antibodies show a dot-like staining pattern that is found in the nuclei and cytoplasm of large neurons of the brainstem and hippocampus (Fig. 12.1d). The Ma protein family concentrates in interchromatin granule clusters and coiled bodies in nuclei and cytoplasm and is involved in transcription and pre-mRNA processing (Rosenfeld et al. 2001). Anti–PKCγ (protein kinase Cγ), anti–ARHGAP26 (rhoGTPase-activating protein 26), and anti-CARPVIII antibodies (carbonic anhydrase-related protein VIII) label the cytoplasm, axons, and dendrites of Purkinje cells (Fig. 12.1e). The antibodies recognize the respective intracellular proteins that are all specific for Purkinje cells (Doss et al. 2014; Hoftberger et al. 2013a, 2014). Anti–SOX1 antibodies (anti-glial nuclear antibody, AGNA) label the nuclei of Bergmann glia of the cerebellum (Fig. 12.1f). SOX1 belongs to a family of transcription factors that is expressed in the developing brain (Graus et al. 2005). Anti–ZIC–antibodies (zinc-finger proteins) label the granule cell layer of the cerebellum and less intensively the cytoplasm of Purkinje cells. ZIC proteins play a role in cerebellar development, and the antibodies target a conserved zinc-finger domain that is common in different ZIC proteins (Bataller et al. 2002). Anti–GAD65 antibodies (glutamic acid decarboxylase 65 antibodies) label axonal terminals in the molecular layer, at the base of Purkinje cells, and rosettes of axonal terminals in the glomeruli of the granular layer of the cerebellum (Fig. 12.1g) (Solimena et al. 1990). GAD65 is the brain-specific isoform of GAD and the rate-limiting enzyme for the synthesis of the transmitter gamma-aminobutyric acid (GABA). Anti–Homer3 antibodies show an intense labeling of the molecular layer of the cerebellum and weaker reactivity with the cytoplasm of Purkinje cells. Homer 3 interacts with the metabotropic glutamate receptor type 1 (mGluR1) and enables clustering of the receptor (Hoftberger et al. 2013c). Anti–adenylate kinase 5 (AK5) antibodies react with the cytoplasm of neurons in cerebrum, cerebellum, and brain stem; nuclei are spared. The protein AK5 is neuron specific and involved in metabolic processes and RNA/DNA synthesis (Tuzun et al. 2007). Anti–Tr antibodies label the Purkinje cell cytoplasm and show a dot-like staining pattern in the molecular layer of the cerebellum. The antigen was initially described intracellular but was recently identified as delta/notch-like epidermal growth factor-related receptor (DNER) (De Graaff et al. 2012).

The association of antibodies targeting surface antigens with malignancy is less consistent. The detection of these antibodies is important because patients usually respond to immunotherapy. Both serum and CSF should be tested, and it is reasonable to follow an algorithmic diagnostic approach (Fig. 12.2):

A first screening should be performed by a tissue-based assay optimized for surface receptor antibodies, where most of the antibodies show a characteristic neuropil staining pattern in the hippocampus. In case of a positive result, the sample should be tested on a cell-based assay that specifically expresses the antigen of interest (e.g., NMDAR, GABA(B)R, AMPAR). If all currently available cell-based assays remain negative, the sample should be stained on live hippocampal neurons. A positive result confirms that the patient’s antibody recognizes a surface receptor antigen, and the technique can be used for characterizing the novel antibody by immunoprecipitation. Glycinreceptor (GlyR) antibodies, dopamin2 receptor (D2R) antibodies, and P/Q type calcium channel (VGCC) antibodies are not detectable by immunohistochemistry and have to be tested either directly by a cell-based assay (GlyR antibodies, D2R antibodies) or by radioimmunoassay (VGCC antibodies).

As mentioned above, various anti-neuronal antibodies have been described in the past few years. In contrast, the role of antibodies to glial antigens for the pathogenesis and diagnosis of inflammatory demyelinating CNS diseases is still unclear, except for antibodies to astrocytic aquaporin-4 (AQP4) in NMO. Evidence for a role of antibodies and B-cells in demyelinating diseases comes from neuropathological investigations (Lassmann et al. 2007), the effect of B-cell directed therapies (Hauser et al. 2008; Keegan et al. 2005; Krumbholz et al. 2012) and the finding of intrathecal immunoglobulin (Ig)G antibody production, dominance of B-cells and oligoclonal IgG bands in the CSF (Cepok et al. 2001; Freedman et al. 2005; Krumbholz et al. 2012; Kuenz et al. 2008; Reindl et al. 2006). Various myelin and non-myelin antigens were suspected as targets for humoral immune reactions in demyelinating diseases (Krumbholz et al. 2012; Reindl et al. 2006). Recently, antibodies to aquaporin-4 (AQP4) have emerged as sensitive and specific biomarkers for NMO (Wingerchuk et al. 2007), and antibodies to the myelin oligodendrocyte glycoprotein (MOG) are associated with a subset of predominantly pediatric demyelinating diseases (Reindl et al. 2013).